Jucara and Açai fruit-based dietary supplements

ABSTRACT

The present disclosure relates to stable, palatable, freeze-dried, fruit-based dietary supplements. In one embodiment, the disclosures relates to compositions of Açai fruit and Jucara fruit with high antioxidant capability and cyclooxygenase-inhibitory activity, and their uses. The disclosure further provides for methods of making stable, palatable, freeze-dried, fruit-based dietary supplements from Açai fruit and Jucara fruit.

RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.10/550,502, filed Jun. 16, 2006, which claims priority to National StageApplication Serial No. PCT/US04/08739, filed Mar. 22, 2004, which claimsthe benefit of and priority to U.S. Application Ser. No. 60/456,882,filed Mar. 21, 2003, the contents of all of which are incorporated byreference herein in their entireties.

BACKGROUND

1. Field

The present disclosure relates to methods of making stable, palatable,freeze-dried, fruit-based dietary supplements, and uses thereof.

2. General Background

Over the past few decades, free radicals have come to be appreciatedincreasingly for their importance to human health and disease. Manycommon and life-threatening diseases, including atherosclerosis, cancer,and aging, have free radical reactions as an underlying mechanism ofinjury. Over this period of time, our conceptual understanding of theinteraction of free radicals with living organisms has evolved andprovided unprecedented opportunities for improving the quality and evenlength of human life.

One of the most common types of free radicals is the reactive oxygenspecies (ROS). These are the products of normal cell respiration andmetabolism and are generally regulated by antioxidants produced in thebody. Due to environmental agents such as pollution, and lifestylefactors such as smoking or exercising, the production of free radicalsis increased. Such increase may bring the body out of balance,especially as the body ages and the mechanisms that produce antioxidantslose their ability to produce these compounds at their necessary rate,resulting in oxidative stress. The resulting damage can range fromdisruption of biological processes, killing of cells, and mutation ofgenetic material, which may lead to the occurrence of cancer.

The potential use of dietary supplements for protection against theeffects of oxidative stress and the progression of degenerative diseasesand aging has been the subject of an increasing number of studies duringthe past two decades. In the market today there are many products thatcontain antioxidants at various levels. These come in the form of foods,liquids and nutritional supplements. The richest sources of these vitalnutrients commonly are found in fruits and vegetables having compoundssuch as Vitamin C, Vitamin E, beta-carotene and others.

The antioxidant hypothesis postulates that supplementation with dietaryantioxidants can alleviate the redox imbalance associated with disease.Antioxidants function to bind these free radicals and stabilize andscavenge them out of the system, thereby reducing the amount of damagefree radicals may cause.

Synthetic antioxidants such as BHA (butylated hydroxy anisole), BHT(butylated hydroxy toluene) and NDGA (nordihydro-guaiaretic acid) havebeen developed to date. By way of examples of natural antioxidants,there are antioxidant enzymes such as superoxide dismutase, peroxidase,catalase and glutathione peroxidase, and non-enzymatic antioxidantsubstances such as tocopherol (vitamin E), ascorbic acid (vitamin C),cartenoid and glutathione.

However, synthetic antioxidants may cause allergic reactions andoncogenesis due to their strong toxicity in the body, and be easilydisrupted by heat due to temperature sensitivity. On the other hand,natural antioxidants are safer than synthetic antioxidants in the bodybut have the problem of weak effect. Therefore, the development of a newnatural antioxidant having no problem with safety in use and also havingexcellent antioxidant activity has been required.

Many studies have demonstrated the protective properties of thepolyphenolic flavonoids. Antimutagenic, anticarcinogenic and immunestimulating properties of flavonoids have been reported. The flavonoidsare a large group of naturally occurring polyphenols found in fruits,vegetables, grains, bark, tea and wine that have proven in vitrofree-radical scavenging potential.

Anthocyanins are naturally occurring compounds that are responsible forthe red, purple, and blue colors of many fruits, vegetables, cerealgrains, and flowers. For example, the colors of berry fruits, such asblueberries, bilberries, strawberries, raspberries, boysenberries,Marion berries, cranberries, are due to many different anthocyanins.Over 300 structurally distinct anthocyanins have been identified innature. Because anthocyanins are naturally occurring, they haveattracted much interest for use as colorants for foods and beverages.Proanthocyanins are another class of flavonoid compounds that are foundin fruits and vegetables and, while being colorless, have antioxidantactivities.

Recently, the interest in anthocyanin pigments has intensified becauseof their possible health benefits as dietary antioxidants. For example,anthocyanin pigments of bilberries (Vaccinium myrtillus) have long beenused for improving visual acuity and treating circulatory disorders.There is experimental evidence that certain anthocyanins and flavonoidshave anti-inflammatory properties. In addition, there are reports thatorally administered anthocyanins are beneficial for treating diabetesand ulcers and may have antiviral and antimicrobial activities. Thechemical basis for these desirable properties of flavonoids is believedto be related to their antioxidant capacity. Thus, the antioxidantcharacteristics associated with berries and other fruits and vegetableshave been attributed to their anthocyanin content.

In the market today there are many products that contain antioxidants atvarious levels. These come in the form of foods, liquids and nutritionalsupplements. The richest sources of these vital nutrients commonly arefound in fruits and vegetables having compounds such as Vitamin C,Vitamin E, anthocyanins, beta-carotene, and others. Antioxidantsfunction to bind these free radicals and stabilize and scavenge them outof the system, thereby reducing the amount of damage free radicals maycause.

Since many fruits and vegetables contain these vital nutrients, it isvery important to be able to assess the ability of antioxidants in thesefoods to absorb free radicals. USDA Researchers at Tufts Universitydeveloped a laboratory test know as ORAC (Oxygen Radical AbsorbanceCapacity) which rates different foods according to their antioxidantcontent and its ability to bind these free radicals. Through this test,different foods may be compared and analyzed for their antioxidantability.

There is a need for the identification of fruits or vegetables with highORAC scores and the development and production of dietary supplementsbased thereon.

SUMMARY

The present disclosure relates to the identification of Açai fruit andJucara fruit with high ORAC scores and cyclooxygenase-inhibitoryactivity. In one aspect the present disclosure provides for a dietarysupplement composition comprising freeze-dried fruit pulp wherein thetotal anthocyanin concentration is greater than about 1 milligram pergram total weight, the composition has an ORAC_(FL) value greater thanabout 350 micromole TE per gram total weight and a residual watercontent less than about 3 percent of the total weight. In oneembodiment, the freeze-dried fruit pulp of the dietary supplementcomposition is freeze-dried Açai fruit pulp. In another embodiment, thefreeze-dried fruit pulp of the dietary supplement is freeze-dried Jucarafruit pulp. In one embodiment the dietary supplement composition of thedisclosure further comprises a pharmaceutically acceptable carrier. In apreferred embodiment, the total anthocyanin concentration of the dietarysupplement composition of the disclosure is from about 1 milligram pergram total weight to about 500 milligram per gram total weight. Inanother preferred embodiment, the total anthocyanin concentration of thedietary supplement is from about 1 milligram per gram to about 100milligram per gram total weight. In yet another preferred embodiment thetotal anthocyanin concentration of the dietary supplement composition isfrom about 1 milligram per gram to about 10 milligram per gram totalweight. In another preferred embodiment, the dietary supplementcomposition has an ORAC_(FL) value from about 350 micromole TE per gramtotal weight to about 10 millimole TE per gram. In another preferredembodiment, the dietary supplement composition has an ORAC_(FL) valuefrom about 350 micromole TE per gram total weight to about 5 millimoleTE per gram. In yet another preferred embodiment, the dietary supplementcomposition has an ORAC_(FL) value from about 350 micromole TE per gramtotal weight to about 1 millimole TE per gram. In a preferredembodiment, the residual water content of the dietary supplementcomposition is from about 0.01 percent to about 3 percent of the totalweight. In another preferred embodiment, the residual water content ofthe dietary supplement composition is from about 0.1 percent to about 3percent of the total weight. In yet another preferred embodiment, theresidual water content of the dietary supplement composition is fromabout 1 percent to about 3 percent of the total weight.

In another aspect, the present disclosure provides for a dietarysupplement composition comprising freeze-dried fruit pulp wherein thecomposition has a cyclooxygenase inhibition value greater than about 15Aspirin® mg equivalent per gram total weight and a residual watercontent less than about 3 weight percent of the total weight. In oneembodiment, the freeze-dried fruit pulp of the dietary supplementcomposition is freeze-dried Açai fruit pulp. In another embodiment, thefreeze-dried fruit pulp of the dietary supplement is freeze-dried Jucarafruit pulp. In one embodiment the dietary supplement composition of thedisclosure further comprises a pharmaceutically acceptable carrier. In apreferred embodiment, the cyclooxygenase inhibition value of the dietarysupplement composition is from about 15 Aspirin® mg equivalent per gramtotal weight to about 10,000 Aspirin® mg equivalent per gram totalweight. In another preferred embodiment, the cyclooxygenase inhibitionvalue of the dietary supplement composition is from about 15 Aspirin® mgequivalent per gram total weight to about 1,000 Aspirin® mg equivalentper gram total weight. In yet another preferred embodiment, thecyclooxygenase inhibition value of the dietary supplement composition isfrom about 15 Aspirin® mg equivalent per gram total weight to about 100Aspirin® mg equivalent per gram total weight. In a preferred embodiment,the residual water content of the dietary supplement composition is fromabout 0.01 percent to about 3 percent of the total weight. In anotherpreferred embodiment, the residual water content of the dietarysupplement composition is from about 0.1 percent to about 3 percent ofthe total weight. In yet another preferred embodiment, the residualwater content of the dietary supplement composition is from about 1percent to about 3 percent of the total weight.

In another aspect the disclosure provides for a method of producing astable and palatable fruit-based dietary supplement composition,comprising harvesting the fruits; weighing the fruits; cleaning thefruits with water; washing the fruits with water at a temperature about75 degrees C. to 100 degrees C. for a period of time of about 5 secondsto 10 minutes; hulling the fruits to isolate the fruit pulp from thefruit; freezing the fruit pulp to a temperature below about −5 degreesC.; and freeze-drying the fruit pulp under conditions to yield agranular, freeze-dried pulp powder with residual water content of lessthan 3 weight percent wherein the freeze-dried fruit pulp powder is morestable and palatable than an fruit pulp preparation. In one embodiment,the fruit is Açai fruit. In another embodiment, the fruit is Jucarafruit. In one embodiment, the cleaning step consists of cleaning thefruits with hygienic water at 0.1% (v/v). In another embodiment, citricacid is added to the fruit pulp preparation prior to freezing. Inanother embodiment, the washing step consists of washing the fruits inwater at a temperature of about 80 degrees C. for a period of time ofabout 10 seconds. In yet another embodiment, the hulling step consistsof mechanically hulling the fruits for a time period of between about 2minutes to 5 about minutes and the hulling step is carried out usingabout 1 liter of water per 2 kg of fruits. In yet another embodiment,the method of making the dietary supplement composition yields afruit-based dietary supplement composition that has an ORAC_(FL) valueof greater than about 350 micromole TE per gram total weight. In anotherpreferred embodiment, the method of making the dietary supplementcomposition yields a fruit-based dietary supplement composition that hasan ORAC_(FL) value from about 350 micromole TE per gram total weight toabout 10 millimole TE per gram. In another preferred embodiment, themethod of making the dietary supplement composition yields a fruit-baseddietary supplement composition that has an ORAC_(FL) value from about350 micromole TE per gram total weight to about 5 millimole TE per gram.In yet another preferred embodiment, the method of making the dietarysupplement composition yields a fruit-based dietary supplementcomposition that has an ORAC_(FL) value from about 350 micromole TE pergram total weight to about 1 millimole TE per gram. In another preferredembodiment, the method of making the dietary supplement compositionyields a fruit-based dietary supplement composition that has acyclooxygenase inhibition value greater than about 15 Aspirin® mgequivalent per gram total weight. In a preferred embodiment, the methodof making the dietary supplement composition yields a fruit-baseddietary supplement composition that has a cyclooxygenase inhibitionvalue from about 15 Aspirin® mg equivalent per gram total weight toabout 10,000 Aspirin® mg equivalent per gram total weight. In anotherpreferred embodiment, the method of making the dietary supplementcomposition yields a fruit-based dietary supplement composition that hasa cyclooxygenase inhibition value from about 15 Aspirin® mg equivalentper gram total weight to about 1,000 Aspirin® mg equivalent per gramtotal weight. In yet another preferred embodiment, the method of makingthe dietary supplement composition yields a fruit-based dietarysupplement composition that has a cyclooxygenase inhibition value fromabout 15 Aspirin® mg equivalent per gram total weight to about 100Aspirin® mg equivalent per gram total weight.

In yet another aspect, the disclosure provides a method of preventing ortreating a disease or an injury induced by pathological free radicalreactions in a mammal, the method comprising administering to the mammalan effective amount of a fruit-based dietary supplement composition ofthe disclosure, wherein the composition quenches free radicals andreduces the damage induced by pathological free radicals. In oneembodiment, the disease or injury is selected from the group consistingof: cancer, colon cancer, breast cancer, inflammatory bowel disease,Crohn's disease, vascular disease, arthritis, ulcer, acute respiratorydistress syndrome, ischemia-reperfusion injury, neurodegenerativedisorders, autism, Parkinson's Disease, Alzheimer's Disease,gastrointestinal disease, tissue injury induced by inflammation, andtissue injury induced by an environmental toxin.

In yet another aspect, the present disclosure provides a method foralleviating the deleterious effects of pathological free radicalreactions in a mammal afflicted with a disease or an injury induced bypathological free radical reactions in a mammal, the method comprisingadministering to the mammal an effective amount of a fruit-based dietarysupplement composition of the disclosure, wherein the compositionquenches free radicals and reduces the damage induced by pathologicalfree radicals. In one embodiment, the disease or injury is selected fromthe group consisting of: cancer, colon cancer, breast cancer,inflammatory bowel disease, Crohn's disease, vascular disease,arthritis, ulcer, acute respiratory distress syndrome,ischemia-reperfusion injury, neurodegenerative disorders, autism,Parkinson's Disease, Alzheimer's Disease, gastrointestinal disease,tissue injury induced by inflammation, and tissue injury induced by anenvironmental toxin.

In yet another aspect, the present disclosure provides a method ofinhibiting cyclooxygenase enzyme activity in a mammal, the methodcomprising administering to the mammal an effective amount of acomposition comprising a fruit-based dietary supplement composition ofthe disclosure. In one embodiment, the fruit-based dietary supplementcomposition further comprises a pharmaceutically acceptable carrier. Inanother embodiment, the a fruit-based dietary supplement composition isadministered by a route of administration selected from the groupconsisting of: oral, intravenous, intraperitoneal, subcutaneous,intramuscular, intraarticular, intraarterial, intracerebral,intracerebellar, intrabronchial, intrathecal, topical, and aerosolroute.

In another aspect, the present disclosure provides a method ofpreventing or treating a disease or an injury associated with increasedcyclooxygenase enzyme activity in a mammal, the method comprisingadministering to the mammal an effective amount of a compositioncomprising the fruit-based dietary supplement composition of thedisclosure. In one embodiment, the fruit-based dietary supplementcomposition further comprises a pharmaceutically acceptable carrier. Inanother embodiment, the fruit-based dietary supplement composition isadministered by a route of administration selected from the groupconsisting of: oral, intravenous, intraperitoneal, subcutaneous,intramuscular, intraarticular, intraarterial, intracerebral,intracerebellar, intrabronchial, intrathecal, topical, and aerosolroute. In another embodiment, the disease or injury is selected from thegroup consisting of: cancer, colon cancer, breast cancer, inflammatorybowel disease, Crohn's disease, vascular disease, arthritis, ulcer,acute respiratory distress syndrome, ischemia-reperfusion injury,neurodegenerative disorders, autism, Parkinson's Disease, Alzheimer'sDisease, gastrointestinal disease, tissue injury induced byinflammation, and tissue injury induced by an environmental toxin.

These and other objects of the present disclosure will be apparent fromthe detailed description of the disclosure provided below.

DRAWINGS

The disclosure will be more fully understood by reference to thefollowing drawings which are for illustrative purposes only:

FIG. 1 is a graph showing a representative absorption spectrum offreeze-dried Açai powder.

FIG. 2 is a graph showing the anthocyanin profile of freeze-dried Jucarapowder as determined by LC/MS/MS chromatographic technique.

FIG. 3 is a schematic diagram showing the chemical structures ofanthocyanins in freeze-dried Jucara powder.

FIG. 4 is a graph showing the anthocyanin profile of freeze-dried Açaipowder as determined by LC/MS/MS chromatographic technique.

FIG. 5 is a schematic diagram showing the chemical structures ofanthocyanins in freeze-dried Açai powder.

FIG. 6 is a graph showing the phenolic compound profile of freeze-driedJucara powder as determined by HPLC and mass spectroscopychromatographic technique.

FIG. 7 is a schematic diagram showing the chemical structures ofphenolic compounds in freeze-dried Jucara powder.

FIG. 8 is a graph showing the proanthocyanin profiles of freeze-driedAçai powder and freeze-dried Jucara powder as determined bychromatographic technique.

FIG. 9 is a schematic diagram showing the chemical structures ofproanthocyanin compound in freeze-dried Açai powder and freeze-driedJucara powder.

FIG. 10 is a histogram graph comparing the antioxidant activity ofselect vegetables as determined by ORAC analysis technique.

FIG. 11 is a histogram graph comparing the antioxidant activity selectfresh fruits as determined by ORAC analysis technique.

FIG. 12 is a histogram graph comparing the antioxidant activity ofselect fresh fruits as determined by ORAC analysis technique.

FIG. 13 is a histogram graph comparing the antioxidant activity offreeze-dried Açai powder and freeze-dried Jucara powder with selectfresh fruits as determined by ORAC analysis technique.

FIG. 14 is a histogram graph comparing the antioxidant activity offreeze-dried Açai with select fresh fruits as determined by ORACanalysis technique.

FIG. 15 is a histogram graph comparing the antioxidant activity offreeze-dried Açai powder with select fresh vegetables as determined byORAC analysis technique.

FIG. 16 is a histogram graph comparing the antioxidant activity ofselect fruits, vegetables and nuts as determined by ORAC analysistechnique.

FIG. 17 is a histogram graph comparing the antioxidant activity ofselect nuts as determined by ORAC analysis technique.

FIG. 18 is a histogram graph comparing the antioxidant activity ofdehydrated Açai with select dehydrated fruits and vegetables asdetermined by ORAC analysis technique.

FIG. 19 is a histogram graph comparing the antioxidant activity offreeze-dried Açai powder with select fresh vegetables as determined byORAC analysis technique.

FIG. 20 is a histogram graph comparing the antioxidant activity ofdehydrated Açai with select dehydrated fruits and vegetables asdetermined by ORAC analysis technique.

FIG. 21 is a histogram graph comparing the antioxidant activity offruits and vegetables by ORAC_(HO) analysis technique.

FIG. 22 is a flow chart schematic diagram detailing Açai fruit juicepreparation.

FIG. 23 is a schematic diagram of the hulling apparatus used in Açaifruit juice preparation.

FIG. 24 is a flow chart schematic diagram detailing a method ofpreparing freeze-dried Açai powder.

DETAILED DESCRIPTION

It is to be appreciated therefore that certain aspects, modes,embodiments, variations and features of the disclosure described belowin various levels of detail in order to provide a substantialunderstanding of the present disclosure. In general, such disclosureprovides beneficial dietary supplement compositions, combinations ofsuch compositions with other dietary supplement compositions, andrelated methods of producing and using same.

Accordingly, the various aspects of the present disclosure relate totherapeutic or prophylactic uses of certain particular dietarysupplement compositions in order to prevent or treat a disease or aninjury induced by pathological free radical reactions. The variousaspects of the present disclosure further relate to therapeutic orprophylactic uses of certain particular dietary supplement compositionsin order to prevent or treat a disease or an injury associated withincreased cyclooxygenase enzyme activity. Accordingly, variousparticular embodiments that illustrate these aspects follow.

It is to be appreciated that the various modes of treatment orprevention of medical conditions as described are intended to mean“substantial”, which includes total but also less than total treatmentor prevention, and wherein some biologically or medically relevantresult is achieved.

Definitions

A “subject,” as used herein, is preferably a mammal, such as a human,but can also be an animal, e.g., domestic animals (e.g., dogs, cats andthe like), farm animals (e.g., cows, sheep, pigs, horses and the like)and laboratory animals (e.g., rats, mice, guinea pigs and the like).

An “effective amount” of a compound, as used herein, is a quantitysufficient to achieve a desired therapeutic and/or prophylactic effect,for example, an amount which results in the prevention of or a decreasein the symptoms associated with a disease that is being treated. Theamount of compound administered to the subject will depend on the typeand severity of the disease and on the characteristics of theindividual, such as general health, age, sex, body weight and toleranceto drugs. It will also depend on the degree, severity and type ofdisease. The skilled artisan will be able to determine appropriatedosages depending on these and other factors. Typically, an effectiveamount of the compounds of the present disclosure, sufficient forachieving a therapeutic or prophylactic effect, range from about0.000001 mg per kilogram body weight per day to about 10,000 mg perkilogram body weight per day. Preferably, the dosage ranges are fromabout 0.0001 mg per kilogram body weight per day to about 100 mg perkilogram body weight per day. The compounds of the present disclosurecan also be administered in combination with each other, or with one ormore additional therapeutic compounds.

“Açai” is a well-known species of palm tree characteristic of thenorthern region of Brazil known as Para. The Açai is characterized by athin trunk and round egg-shaped clustered fruits that are dark purple,sometimes even verging on black when ripe. The Latin name for Açai isEuterpe oleracea, Martius; family, Palmaceae. It is also known inEnglish as “Cabbage Palm.” In Brazil it is known as: acai-do-para,acai-do-baixo Amazonas, palmito acai, acaizeiro, acai, assai, jicara,jucara, palmiteiro, piria; in Colombia it is known as: assai and manaca;and uacai; and in Surinam it is known as: manaka, pinapalm, prasara,wapoe, and wasei. The term Açai also includes another Euterpesub-species, E. catinga Wallace, which also found in Brazil and referredto as “açai”. Finally, term Açai also includes another Euterpesubspecies, E. precatoria Martius, which is found in Bolivia and knownto the South American regions and also called “açai” and “jucara”.

“Jucara” is another species of palm tree. The Latin name for jucara is:Euterpe edulis, Martius; family, Palmaceae. It is also known in Brazilas: assai, acai, plamito, palmito doce, iucara, palmito jucara, ripeira,icara, jucara, ensarova, palmiteiro. The term Jucara also includesanother Euterpe sub-species, E. espiritosantensis Fernandes, which alsofound in Brazil, referred to as “jucara”. Finally, the term Açai alsoincludes another Euterpe subspecies, E. precatoria Martius, which isfound in Bolivia and known to the South American regions and also called“acai” and “jucara”.

The references cited throughout this application are incorporated hereinby reference in their entireties.

Antioxidant Properties and Uses Thereof

The present disclosure identified the fruits of two families of palmtrees, Açai and jucara, as having ORAC scores significantly higher thanany other fruits or vegetables tested.

The Açai fruits were known to contain a high proportion ofmono-unsaturated and polyunsaturated fatty acids, and a relatively lowconcentration of saturated fat and trans fatty acids. The Açai fruitswere also known to be rich in lipids, fibers and protein, and to containVitamin E and anthocyanins, two known antioxidants. However, thesefruits have been underutilized in the past because the Açai fruits arevery prone to rapid deterioration due to oxidation and microbialcontamination by bacteria, fungi and yeast. Accordingly, the fruit andjuice made from the Açai fruits deteriorate rapidly, and quickly losetheir palatability and antioxidant properties—almost half of theanthocyanins degrade within two days after the fruit is picked. In aneffort to overcome the rapid deterioration of Açai fruit and juice, andthereby expose the product to broader markets, some companies have triedfreezing the fruit pulp. However, simply freezing the Açai fruit pulp inthis manner requires careful monitoring of the temperature—with evenrelatively slight deviations in temperature resulting in the activationof deteriorating enzymes and fermenting agents. Moreover, when thawingsuch frozen fruit pulp for use, these agents also become activatedresulting in grittiness to the pulp.

The foregoing problems, among others, have been resolved by the presentdisclosure. Specifically, as described in the Examples below, thepresent disclosure provides a stable and palatable Açai-based dietarysupplement composition with significantly higher anthocyaninconcentration and higher ORAC scores than any other freeze-dried fruitor vegetable compositions tested.

As a result of the present disclosure, it is now apparent that the Açaifruit provides a very good source for a dietary supplement. Prior to thepresent disclosure, the fruit was used primarily as an energy drink oras part of a frozen treat with a short shelf life. The Açai-baseddietary supplement compositions of the present disclosure provide astable and palatable product that has a significantly longer shelf life,while significantly increasing the antioxidant properties of the Açaifruit. The present disclosure allows the highly nutritious features ofthe fruit to not only be preserved, but to be significantly enhanced,and to be enjoyed without the associated concerns of rapid degradation.

While the foregoing discussion focuses primarily on the Açai fruit anddietary supplements derived therefrom, the present disclosure alsoprovide Jucara-based dietary supplement compositions that also containsignificantly higher anthocyanin concentration and produced higher ORACscores than any other freeze-dried fruit or vegetable compositionstested. As will be described below, the Jucara fruit, and dietarysupplements derived therefrom, were also found to very high levels ofproanthocyanidins and exhibited high antioxidant activities againsthydroxy radical and peroxynitrite.

According to the present disclosure, the Açai fruit and the Jucarafruit, juice, dietary supplements, and other compositions derived fromthe Açai fruit and the Jucara fruit be used to treat, reverse, and/orprotect against the deleterious effects of free radicals and oxidativestress.

Free Radicals and Oxidative Stress

Over the past few decades, free radicals, highly reactive and therebydestructive molecules, have come to be appreciated increasingly fortheir importance to human health and disease. Many common andlife-threatening human diseases, including atherosclerosis, cancer, andaging, have free radical reactions as an underlying mechanism of injury.

A free radical is a molecule with one or more unpaired electrons in itsouter orbital. Many of these molecular species are oxygen (and sometimesnitrogen) centered. Indeed, the molecular oxygen we breathe is a freeradical. These highly unstable molecules tend to react rapidly withadjacent molecules, donating, abstracting, or even sharing their outerorbital electron(s). This reaction not only changes the adjacent, targetmolecule, sometimes in profound ways, but often passes the unpairedelectron along to the target, generating a second free radical or otherROS, which can then go on to react with a new target. In fact, much ofthe high reactivity of ROS is due to their generation of such molecularchain reactions, effectively amplifying their effects many fold.Antioxidants afford protection because they can scavenge ROS before theycause damage to the various biological molecules, or prevent oxidativedamage from spreading, e.g., by interrupting the radical chain reactionof lipid peroxidation.

ROS and Human Health

Because our bodies are continuously exposed to free radicals and otherROS, from both external sources (sunlight, other forms of radiation,pollution) and generated endogenously, ROS-mediated tissue injury is afinal common pathway for a number of disease processes.

Radiation Injury

Radiation injury represents an important cause of ROS-mediated disease.Extreme examples include the physical-chemical reactions within thecenter of the sun and at the center of a thermonuclear blast. Withrespect to more commonly encountered levels of radiation, depending uponthe situation, about two-thirds of the sustained injury is mediated notby the radiation itself, but by the ROS generated secondarily. Thisapplies not only to the acutely toxic forms of radiation injury, but thelong-term, mutagenic (and hence carcinogenic) effects as well.

An important clinical application of this principle is encounteredregularly in the treatment of cancer by radiation therapy. Large tumorsoften outgrow their blood supplies and tumor cells die within thecenter, despite being well oxygenated at the periphery. Between thesetwo regions is an area of tumor that is poorly oxygenated, yet remainsviable. Radiation therapy of such tumors is particularly effective atthe periphery, where an abundant concentration of oxygen is available toform tumorcidal ROS. The poorly oxygenated center is injured to asignificantly smaller degree. While the dead cells in the center don'tsurvive anyway, the poorly oxygenated, yet viable, cells between thesetwo areas can survive a safe dose of radiation therapy, and thereby seeda later local recurrence of the tumor. This is a major reason why manylarge tumors are treated by a combination of radiation therapy (to killthe tumor at its advancing edges) and surgical removal of the bulk ofthe tumor, including these particularly dangerous remaining cells.

Cancer and Other Malignancies

Cancer and other malignancies all entail unconstrained cell growth andproliferation based upon changes in the cell's genetic information. Inmost cases, for example, one or more genes that normally constrain cellgrowth and replication is/are mutated or otherwise inactivated. Thesegenetic deficiencies correspond directly with deletions and sequencechanges in the genetic code, resident in the cell's DNA. A frequentlyseen final common cause of such DNA damage is free radical injury. Ofthe myriad injuries sustained by our DNA on a daily basis, most arerepaired by normal DNA repair mechanisms within the cell, while someresult in cell death. Since such injuries are sporadic and distributedsomewhat randomly across the genome, most lethal DNA injuries areclinically inconsequential, resulting in the loss of a few cells amongmillions. However, when a single cell sustains an injury that impairsgrowth regulation, it can proliferate disproportionately and growrapidly to dominate the cell population by positive natural selection.The result is a tumor, frequently a malignant one, where the constraintof growth and proliferation is particularly deficient. Therefore, freeradical injury to the genetic material is a major final common pathwayfor carcinogenesis.

ROS can be generated within the cell not only by external sources ofradiation, but also within the body as a byproduct of normal metabolicprocesses. An important source of endogenous free radicals is themetabolism of some drugs, pollutants, and other chemicals and toxins,collectively termed xenobiotics. While some of these are directly toxic,many others generate massive free radical fluxes via the very metabolicprocesses that the body uses to detoxify them. One example is themetabolism of the herbicide paraquat. At one time, drug enforcementauthorities used this herbicide to kill marijuana plants. Growersrealized they could harvest the sprayed crop before it wilted, and stillsell the paraquatlaced product. Many who smoked this productsubsequently died of a fulminant lung injury. Fortunately, this approachhas been abandoned as a particularly inhumane way to solve the drugproblem.

While the paraquat story is a particularly striking example of ametabolic mechanism of free radical toxicity, many commonly encounteredxenobiotics, including cigarette smoke, air pollutants, and even alcoholare toxic, and often carcinogenic to a large degree by virtue of thefree radicals generated by their catabolism within our bodies. Moreover,there is accumulating evidence that a diet rich in fruits andvegetables, which are high in natural antioxidants, and low in saturatedfat (a particularly vulnerable target for damage by ROS), reduces therisk of atherosclerosis and cancer.

Atherosclerosis

Atherosclerosis remains the major cause of death and prematuredisability in developed societies. Moreover, current predictionsestimate that by the year 2020 cardiovascular diseases, notablyatherosclerosis, will become the leading global cause of total diseaseburden, defined as the years subtracted from healthy life by disabilityor premature death. Atherosclerosis is a complex process that leads toheart attack, stroke, and limb loss by the plugging of the arteries withatherosclerotic plaque. This plaque is a form of oxidized fat. When freeradicals react with lipids, the consequence is lipid peroxidation, thesame process by which butter turns rancid when exposed to the oxygen inthe air. While a number of factors influence the development andseverity of atherosclerosis, a major factor is the ROS-mediatedperoxidation of our low-density lipoproteins (LDLs, or “badcholesterol”. The dietary approach to the prevention of heart diseaseand stroke is based partially on adding dietary antioxidants to limitLDL oxidation, as well as decreasing the intake of fat itself. Theseapproaches already have made significant inroads into the mortality fromheart disease, but the compositions of the present disclosure may offera safe pharmacological prevention in the future that is not as dependentupon willpower as are diet and exercise.

Neurological and Neurodegenerative Diseases

Neurological and neurodegenerative diseases affect millions ofAmericans. These include depression, obsessive-compulsive disorder,Alzheimer's, allergies, anorexia, schizophrenia, as well as otherneurological conditions resulting from improper modulation ofneurotransmitter levels or improper modulation of immune systemfunctions, as well as behavioral disorders such as ADD (AttentionDeficit Disorder) and ADHD (Attention Deficit Hyperactivity Disorder). Anumber of these diseases appear to have ROS toxicity as a centralcomponent of their underlying mechanism of nerve cell destruction,including, but not limited to, amyotrophic lateral sclerosis (ALS, orLou Gehrig's disease), Parkinson's disease, and Alzheimer's disease.

Ischemia/Reperfusion Injury

When an organ is deprived of its blood supply (ischemia) it is injured,not just by the temporary loss of oxygen, but also by the ROS that aregenerated by reaction with the oxygen that is reintroduced atreperfusion, when the blood supply is restored. In some clinicalsituations, this injury can prevented by giving antioxidants, sometimeseven after the period of ischemia, but just prior to reperfusion. Forexample, the preservation of kidneys, livers, and other organs insolutions that contain antioxidants, as well as other agents, is nowroutine prior to their transplantation. Another example is the use ofdrugs that block the function of free radical generating enzymes priorto stopping the heart for cardiac surgery. These drugs help preventreperfusion injury when the heart is restarted and flow is restored.This reperfusion injury mechanism also has been found to play animportant role in patients suffering from multiple organ failure aftertrauma, massive surgery, or shock. Multiple organ failure is now theleading cause of death in intensive care units, and extensive effortsare under way to understand better how ROS contribute to this syndrome.

Aging

Aging is a remarkably complex process that has managed to remainrelatively opaque to scientific understanding. There is now evidencethat aging is a series of processes, i.e., a series of controlledmechanisms, and not just the passive accumulation of wear and tear overthe years. If aging is a series of processes, some of these processesare potentially controllable, or at least modifiable. One of the mostimportant of these processes is comprised of an accumulation of themolecular injuries that are mediated by free radicals and other ROS.Recent studies indicate that the therapeutic manipulation of ROSmetabolism can actually extend the total life span of mice to asignificant degree.

Autistic Disorder

Autism is a disabling neurological disorder that affects thousands ofAmericans and encompasses a number of subtypes, with various putativecauses and few documented ameliorative treatments. The disorders of theautistic spectrum may be present at birth, or may have later onset, forexample, at ages two or three. There are no clear-cut biological markersfor autism. Diagnosis of the disorder is made by considering the degreeto which the child matches the behavioral syndrome, which ischaracterized by poor communicative abilities, peculiarities in socialand cognitive capacities, and maladaptive behavioral patterns.

A number of different treatments for autism have been developed. Many ofthe treatments, however, address the symptoms of the disease, ratherthan the causes. For example, therapies ranging from psychoanalysis topsychopharmacology have been employed in the treatment of autism.Although some clinical symptoms may be lessened by these treatments,modest improvement, at best, has been demonstrated in a minor fractionof the cases. Only a small percentage of autistic persons become able tofunction as self-sufficient adults.

In a preliminary study, an Açai-based dietary supplement was provided toan autistic child with very limited speech and the child wassubsequently reported to have significantly enhanced speech.

Properties and Uses Cyclooxygenase Inhibitor

The present disclosure identified the fruits of two families of palmtrees, Açai and jucara, as having significant inhibitory properties ofboth isoforms of cyclooxygenase, COX-1 and COX-2. Cyclooxygenases(sometimes called prostaglandin endoperoxide synthase) are involved inprostaglandin synthesis. COX-1 expression is considered to beconstitutive, as basal levels of COX-1 mRNA and protein are observed tobe present and generate prostaglandins for normal physiologicalfunctions. In contrast, COX-2 expression is inducible. According to thepresent disclosure, the Açai fruit and the Jucara fruit, juice, dietarysupplements, and other compositions derived from the Açai fruit and theJucara fruit be used to treat, reverse, and/or prevent diseases orinjuries associated with increased cyclooxygenase activity.

Gastroduodenal Mucosal Defense

The gastric epithelium is under a constant assault by a series ofendogenous noxious factors including HCl, pepsinogen/pepsin, and bilesalts. In addition, a steady flow of exogenous substances such asmedications, alcohol, and bacteria encounter the gastric mucosa. Ahighly intricate biologic system is in place to provide defense frommucosal injury and to repair any injury that may occur.

Prostaglandins play a central role in gastric epithelial defense/repair.The gastric mucosa contains abundant levels of prostaglandins. Thesemetabolites of arachidonic acid regulate the release of mucosalbicarbonate and mucus, inhibit parietal cell secretion, and areimportant in maintaining mucosal blood flow and epithelial cellrestitution. Prostaglandins are derived from esterified arachidonicacid, which is formed from phospholipids (cell membrane) by the actionof phospholipase A₂. A key enzyme that controls the rate-limiting stepin prostaglandin synthesis is cyclooxygenase (COX), which is present intwo isoforms (COX-1, COX-2), each having distinct characteristicsregarding structure, tissue distribution, and expression. COX-1 isexpressed in a host of tissues including the stomach, platelets,kidneys, and endothelial cells. This isoform is expressed in aconstitutive manner and plays an important role in maintaining theintegrity of renal function, platelet aggregation, and gastrointestinalmucosal integrity. In contrast, the expression of COX-2 is inducible byinflammatory stimuli, and it is expressed in macrophages, leukocytes,fibroblasts, and synovial cells. The beneficial effects of nonsteroidalanti-inflammatory drugs (NSAIDs) on tissue inflammation are due toinhibition of COX-2. COX-2-inhibitors have the potential to provide thebeneficial effect of decreasing tissue inflammation while minimizingtoxicity in the gastrointestinal tract.

Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic multisystem disease of unknowncause. Although there are a variety of systemic manifestations, thecharacteristic feature of RA is persistent inflammatory synovitis,usually involving peripheral joints in a symmetric distribution. Thepotential of the synovial inflammation to cause cartilage destructionand bone erosions and subsequent changes in joint integrity is thehallmark of the disease.

The first line of medical management of RA the use of nonsteroidalanti-inflammatory drugs (NSAIDs) and simple analgesics to control thesymptoms and signs of the local inflammatory process. These agents arerapidly effective at mitigating signs and symptoms, but they appear toexert minimal effect on the progression of the disease. NSAIDs block theactivity of the Cox enzymes and therefore the production ofprostaglandins, prostacyclin, and thromboxanes. As a result, they haveanalgesic, anti-inflammatory, and antipyretic properties. In addition,the agents may exert other anti-inflammatory effects. Since these agentsare all associated with a wide spectrum of toxic side effects, thenatural dietary supplement compositions of the present disclosure couldprovide a non-toxic alternative to NSAIDs.

Cancer

Cyclooxygenases have been studied in various cancers, and COX-1 or COX-2appear to have a role in several forms of cancer. For example, bothCOX-1 and COX-2 have been shown to be highly expressed in lung cancer inthe mouse. (Bauer et al., 2000, Carcinogenesis 21, 543-550). COX-1 wasreported to be induced by tobacco carcinogens in human macrophages andis correlated with NFκB activation. (Rioux and Castonguay, 2000,Carcinogenesis 21, 1745-1751). COX-1 but not COX-2 was reported to beexpressed in human ovarian adenocarcinomas. (Dor et al., 1998, J.Histochem. Cytochem. 46, 77-84). According to Ryu et al. (2000,Gynecologic Oncology 76, 320-325), COX-2 expression is high in stage 1Dcervical cancer. COX-2 was reported to be over expressed in humancervical cancer. (Kulkami et al., 2001, Clin. Cancer Res. 7, 429-434).Finally, COX-1 was reported to be upregulated in cervical carcinoma andinhibitors of COX-1 were proposed for the treatment of neoplasticcondition of the cervix. Sales et al., US Patent Application20030220266.

According to the present disclosure, the Açai fruit and the Jucarafruit, juice, dietary supplements, and other compositions derived fromthe Açai fruit and the Jucara fruit be used to treat, reverse, and/orprevent cancers associated with increased cyclooxygenase activity.

Pharmaceutical Compositions and Formulations

The fruit-based dietary supplements of the present disclosure can beused in beverages, tonics, infusions, or foodstuffs alone, or incombination with other dietary supplements or therapeutics. Thefruit-based dietary supplements of the disclosure can be used alone orfurther formulated with pharmaceutically acceptable compounds, vehicles,or adjuvants with a favorable delivery profile, i.e., suitable fordelivery to a subject. Such compositions typically comprise thefruit-based dietary supplement of the disclosure and a pharmaceuticallyacceptable carrier. As used herein, “pharmaceutically acceptablecarrier” is intended to include any and all solvents, dispersion media,coatings, antibacterial and antifungal compounds, isotonic andabsorption delaying compounds, and the like, compatible withpharmaceutical administration. Suitable carriers are described in themost recent edition of Remington's Pharmaceutical Sciences, a standardreference text in the field, which is incorporated herein by reference.Preferred examples of such carriers or diluents include, but are notlimited to, water, saline, Ringer's solutions, dextrose solution, and 5%human serum albumin. Liposomes and non-aqueous vehicles such as fixedoils may also be used. The use of such media and compounds forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or compound is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the disclosure is formulated to becompatible with its intended route of administration. Examples of routesof administration include oral, intravenous, intraperitoneal,subcutaneous, intramuscular, intraarticular, intraarterial,intracerebral, intracerebellar, intrabronchial, intrathecal, topical,and aerosol route. The pH can be adjusted with acids or bases, such ashydrochloric acid or sodium hydroxide.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules, caplets or compressedinto tablets. For the purpose of oral therapeutic administration, thefruit-based dietary supplements of the disclosure can be incorporatedwith excipients and used in the form of tablets, troches, or capsules.Oral compositions can also be prepared using a fluid carrier for use asa mouthwash, wherein the compound in the fluid carrier is applied orallyand swished and expectorated or swallowed. Pharmaceutically compatiblebinding compounds, and/or adjuvant materials can be included as part ofthe composition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegratingcompound such as alginic acid, Primogel, or corn starch; a lubricantsuch as magnesium stearate or Sterotes; a glidant such as colloidalsilicon dioxide; a sweetening compound such as sucrose or saccharin; ora flavoring compound such as peppermint, methyl salicylate, or orangeflavoring.

The fruit-based dietary supplements of the disclosure can also beprepared as pharmaceutical compositions in the form of suppositories(e.g., with conventional suppository bases such as cocoa butter andother glycerides) or retention enemas for rectal delivery.

In one embodiment, the fruit-based dietary supplements of the disclosureare prepared with carriers that will protect the compound against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on the unique characteristics of the fruit-based dietarysupplement and the particular therapeutic effect to be achieved, and thelimitations inherent in the art of compounding such an active compoundfor the treatment of individuals. The pharmaceutical compositions can beincluded in a container, pack, or dispenser together with instructionsfor administration.

The disclosure is further defined by reference to the followingexamples, which are not meant to limit the scope of the presentdisclosure. It will be apparent to those skilled in the art that manymodifications, both to the materials and methods, may be practicedwithout departing from the purpose and interest of the disclosure.

EXAMPLES Example 1 Composition Analysis of Freeze-Dried Açai

Composition analysis of freeze-dried Açai OPTACAI; Lot #: 231003/0410-Cis detailed below in Table 1 and Table 2.

TABLE 1 Specifications Product: Açai powder Appearance Powder (Conforms)Common Name: Açai Color Dark purple Botanical Name: Euterpe oleracea M(Conforms) Botanical Family: Palmae Odor Characteristic Plant Part Used:Frozen Fruit Pulp (Conforms) Harvest Method: Wildcrafted FlavorCharacteristic Identification Method: HPLC (Conforms) Excipient NoneDrying Vacuum Method freeze-dried Mesh size 100% through 80 meshPackaging Plastic & fiberboard Shelf life 2 yrs under proper conditionsMoisture 1% Content Re-hydration 1:13 water Food Analysis ImpuritiesCalories 534 Total heavy metals <10 ppm Calories from fat 292 Lead 22ppb Total fat 32.5 g Pesticide residue Wildcrafted Saturated fat 8.1 gSolvent residue None Cholesterol 13.5 mg Sodium 30.4 mg Totalcarbohydrate 52.2 g Fiber 44.2 g Sugars 1.3 g Protein 8.1 g Moisture 3.4g Ash 3.8 g Microbiology Total aerobic bacterial count <10,000 CFU/gTotal fungal count (mold/yeast) 440 Escherichia coli (45° C./g) AbsentSalmonella Absent Staphylococcus Absent

TABLE 2 ANALYTE RESULT/UNIT UNIT/GRAM Beta carotene 34,800 IU 348 IUVitamin C (ascorbate ion) 1,183 mg 11.83 mg Vitamin E (d-alphatocopherol) 648 IU 6.48 IU Vitamin D 1,252 IU 12.52 IU Vitamin Bi(thiamin) 17.5 mg 0.175 mg Vitamin B2 (riboflavin) 22.9 mg 0.229 mgVitamin B3 (niacin/niacinamide) 129.1 mg 1.291 mg Vitamin B6(pyridoxine) 31.9 mg 0.319 mg Folic acid 600 mcg 0.006 mg Vitamin B12(cyanocobalamin) 400 mcg 0.004 mg Biotin 1.8 mg 0.006 mg Inositol 254.2mg 2.452 mg Calcium 55.1 mg 0.551 mg Iron 0.1 mg 0.001 mg Iodine 700 mcg0.007 mg Magnesium 730 mg 7.302 mg Zinc 0.6 mg 0.006 mg Selenium 200 mcg0.002 mg Copper 500 mcg 0.005 mg Manganese 19 mg 0.190 mg Chromium 6200mcg 0.062 mg Molybdenum 0.00 mg 0.000 mg Potassium 3310 mg 33.10 mgBoron 5.6 mg 0.056 mg Heavy Metal Result Lead (Pb) 22.0 ppb

Unless otherwise specified, all methods were performed as described inthe Official Methods of Analysis of AOAC International, 17th Edition,2000 (hereinafter, AOAC). Moisture content of test sample was measuredusing AOAC method reference #926.08. Protein content of test sample wasmeasured using AOAC method reference #991.20E. Fat content of testsample was measured using AOAC method reference #933.05. Ash content oftest sample was measured using AOAC method reference #935.42.Carbohydrate content of test sample was calculated by difference.Caloric content of test sample was calculated using Atwarter Factors.Sugars were measured using AOAC method reference #982.14. Total dietaryfiber was measured in test sample using AOAC method reference #991.43.Cholesterol content of test sample was measured using AOAC methodreference #994.10. The fatty acid profile of test sample was measuredusing AOAC method reference #969.33. The sodium, calcium and ironcontent of test sample was measured using AOAC method reference #984.27.The vitamin C content of test sample was measured using AOAC methodreference #967.22. The vitamin A content of test sample was measured bythe method of Reynolds and Judds, Analyst, 109:489, 1984.

Microbiological testing was conducted essentially as detailed in Example36 (infra). Trace minerals/metals were analyzed by IPC/MS (AligentHP-7500a) method by IBC Labs (Integrated Biomolecule Corporation,Tucson, Ariz.).

Example 2 Composition Analysis of Freeze-Dried Açai

Composition analysis of freeze-dried Açai FD berry powder(lot#231003/0410-C) was performed by IBC Labs (Integrated BiomoleculeCorporation, Tucson, Ariz.). The results are detailed below in Table 3.

TABLE 3 ANALYTE RESULT UNIT Vitamin A (as beta-carotene) 348 IU/gVitamin C (as ascorbate ion) 11.83 mg/g Vitamin E (as d-alphatocopherol) 6.48 IU/g Vitamin D (as chotecalciferol) 12.52 IU/g VitaminB-1 (as thiamin) 0.175 mg/g Vitamin B-2 (as riboflavin) 0.229 mg/gVitamin B-3 (as niacin/niacinamide) 1.291 mg/g Vitamin &-6 (aspyridoxine) 0.319 mg/g Vitamin B-12 (as cyanocobalamin) 0.004 mg/gPantothenic acid (as free anion) 0.561 mg/g Biotin 0.018 mg/g Folic Add0.006 mg/g Inositol 2.452 mg/g Calcium 0.551 mg/g Magnesium ion 7.302mg/g Copper ion 0.005 mg/g Chromium ion 0.062 mg/g Zinc ion 0.006 mg/gIron ion 0.001 mg/g Sodium ion 0.290 mg/g Manganese ion 0.190 mg/gSelenium ion 0.002 mg/g Boron ion 0.056 mg/g Potassium ion 33.10 mg/gMolybdenum ion 0.000 mg/g Iodine ion 0.007 mg/g Lead ion 22.0 ppb

Unless otherwise specified, all methods were performed as described inthe Official Methods of Analysis of AOAC International, 17th Edition,2000 (hereinafter, AOAC). Moisture content of test sample was measuredusing AOAC method reference #926.08. Protein content of test sample wasmeasured using AOAC method reference #991.20E. Fat content of testsample was measured using AOAC method reference #933.05. Ash content oftest sample was measured using AOAC method reference #935.42.Carbohydrate content of test sample was calculated by difference.Caloric content of test sample was calculated using Atwarter Factors.Sugars were measured using AOAC method reference #982.14. Total dietaryfiber was measured in test sample using AOAC method reference #991.43.Cholesterol content of test sample was measured using AOAC methodreference #994.10. The fatty acid profile of test sample was measuredusing AOAC method reference #969.33. The sodium, calcium and ironcontent of test sample was measured using AOAC method reference #984.27.The vitamin C content of test sample was measured using AOAC methodreference #967.22. The vitamin A content of test sample was measured bythe method of Reynolds and Judds, Analyst, 109:489, 1984. Traceminerals/metals were analyzed by IPC/MS (Aligent HP-7500a) method by IBCLabs (Integrated Biomolecule Corporation, Tucson, Ariz.).

Example 3 Nutritional Analysis of Freeze-Dried Açai

Nutritional analysis for a 10 g serving of freeze-dried Açai wasperformed by Silliker, Inc. Illinois Laboratory (Chicago Heights, Ill.;laboratory ID No. 170547501). The results are detailed below in Table 4.

TABLE 4 ANALYTICAL ANALYTICAL ROUNDED DATA PER DATA PER DATA PER % DAILY100 G SERVING SERVING VALUE LABEL ANALYTES Calories 533.9 533.9 530Calories from Fat 292.6 292.6 290 Total Fat (G) 32.51 32.51 33 51Saturated Fat (G) 8.09 8.09 8 40 Cholesterol (MG) 13.5 13.5 15 5 Sodium(MG) 30.4 30.4 30 1 Total Carbohydrate (G) 52.2 52.2 52 17 Dietary Fiber(G) 44.23 4.23 44 176 Sugars (G) 1.26 1.26 1 Protein (F = 6.25) (G) 8.118.11 8 Vitamin A (IU) 1002 1002 20 Vitamin C (MG) <1.0 <1.0 * Calcium(MG) 260 260 25 Iron (MG) 4.4 4.4 25 CONTRIBUTING ANALYTES Moisture (G)3.39 3.39 Ash (G) 3.78 3.78 Beta Carotene (IU) <5 <5 Retinol (IU) 10021002 Vit. A % Beta Carotene * SUGAR PROFILE Fructose 0.39 Lactose <0.10Sucrose <0.10 Glucose 0.76 Maltose 0.11 * Contains less than 2% of theDaily Value of this nutrient. To calculate the values contained in a 25g serving size, divide all the above values by a factor of 4. A typicalbeverage serving is 25 g.

Unless otherwise specified, all methods were performed as described inthe Official Methods of Analysis of AOAC International, 17th Edition,2000 (hereinafter, AOAC). Moisture content of test sample was measuredusing AOAC method reference #926.08. Protein content of test sample wasmeasured using AOAC method reference #991.20E. Fat content of testsample was measured using AOAC method reference #933.05. Ash content oftest sample was measured using AOAC method reference #935.42.Carbohydrate content of test sample was calculated by difference.Caloric content of test sample was calculated using Atwarter Factors.Sugars were measured using AOAC method reference #982.14. Total dietaryfiber was measured in test sample using AOAC method reference #991.43.Cholesterol content of test sample was measured using AOAC methodreference #994.10. The fatty acid profile of test sample was measuredusing AOAC method reference #969.33. The sodium, calcium and ironcontent of test sample was measured using AOAC method reference #984.27.The vitamin C content of test sample was measured using AOAC methodreference #967.22. The vitamin A content of test sample was measured bythe method of Reynolds and Judds, Analyst, 109:489, 1984.

Example 4 Nutritional Analysis of Freeze-Dried Jucara Fruit

Nutritional analysis for a 100 g serving of freeze-dried Jucara fruitwas performed by Silliker, Inc. Illinois Laboratory (Chicago Heights,Ill.; laboratory ID No. 171378581). The results are detailed below inTable 5.

TABLE 5 ANALYTICAL ANALYTICAL ROUNDED DATA PER DATA PER DATA PER % DAILY100 G SERVING SERVING VALUE LABEL ANALYTES Calories 370.2 370.2 370Calories from Fat 22.4 22.4 20 Total Fat (g) 2.48 2.48 2.5 4 SaturatedFat (g) 0.68 0.68 0.5 2 Cholesterol (mg) <1.0 <1.0 0 0 Sodium (mg) 25.525.5 25 1 Total Carbohydrate (g) 86.3 86.3 86 29 Dietary Fiber (g) 0.830.83 <1 4 Sugars (g) <0.10 <0.10 0 Protein (F = 6.25) (g) 0.68 0.68 <1Vitamin A (IU) 179 179 4 Vitamin C (mg) <1.0 <1.0 * Calcium (mg) 33.033.0 4 Iron (mg) 0.53 0.53 2 CONTRIBUTING ANALYTES Moisture (g) 8.628.62 Ash (g) 1.93 1.93 Beta Carotene IU) 179 179 Retinol (IU) <5 <5 Vit.A % Beta Carotene 100 SUGAR PROFILE Dextrose <0.10 (g/100 g) Lactose<0.10 (g/100 g) Sucrose <0.10 (g/100 g) Fructose <0.10 (g/100 g) Maltose<0.10 (g/100 g) * Contains less than 2% of the Daily Value of thisnutrient.

Unless otherwise specified, all methods were performed as described inthe Official Methods of Analysis of AOAC International, 17th Edition,2000 (hereinafter, AOAC). Moisture content of test sample was measuredusing AOAC method reference #926.08. Protein content of test sample wasmeasured using AOAC method reference #991.20E. Fat content of testsample was measured using AOAC method reference #933.05. Ash content oftest sample was measured using AOAC method reference #935.42.Carbohydrate content of test sample was calculated by difference.Caloric content of test sample was calculated using Atwarter Factors.Sugars were measured using AOAC method reference #982.14. Total dietaryfiber was measured in test sample using AOAC method reference #991.43.Cholesterol content of test sample was measured using AOAC methodreference #994.10. The fatty acid profile of test sample was measuredusing AOAC method reference #969.33. The sodium, calcium and ironcontent of test sample was measured using AOAC method reference #984.27.The vitamin C content of test sample was measured using AOAC methodreference #967.22. The vitamin A content of test sample was measured bythe method of Reynolds and Judds, Analyst, 109:489, 1984.

Example 5 Quantitative Analysis of Sterols in Freeze-Dried Açai Powder

The sterol composition of freeze-dried Açai powder (#001 Açai Powder;Flora ID No. 210823003) was determined by High Resolution GasChromatography (HRGC) (Flora Research Laboratories, Grants Pass, Oreg.)as summarized in Table 6.

TABLE 6 ANALYTE PERCENT BY WEIGHT B-Sitosterol  0.044 = 0.44 mg/gCampesterol <0.003 = 0.3 mg/g Sigmasterol  0.004 = 0.04 mg/g TotalSterols  0.048

Example 6 Analysis of the Residual Humidity Analysis of Freeze-DriedAçai

The residual humidity of Açai preparations were determined before andafter freeze-drying by the method of Instituto Adolfo Lutz (1976)(UNIVERSIDADE DE SÃO PAULO, Faculdade do Clencias FarmaceuticasDepartamento de Alimentos e Nutricillo Experimental Laboratorio deAnaliste de Alimentos). The percent humidity of raw Açai pulp was85.37+/−0.14%. The percent residual humidity of freeze-dried Açai pulpwas 1.06%. The antocianinas totals (mg/100 g Açai pulp) was239.32+/−0.74 as determined by the method of Francis and Fuleki, (J.Food Sci, v. 33, p. 72-77, 1968) FIG. 1 shows a representativeabsorption spectrum observed for Freeze-dried Açai powder.

Example 7 Analysis of Anthocyanins and Phenolic Compounds in Jucara andAçai Preparations

I. General

A. Proanthocyanidins

Proanthocyanidins may help explain the “French Paradox,” or why lowcoronary heart disease rates exist in French provinces known forhigh-fat foods and red wine consumption. Red wine could be considered analcohol tincture of several potent flavonoids, includingproanthocyanidins from grape seeds. In a provocative study, FulvioUrsini, M.D., from the University of Padova, Italy, fed volunteers ahigh-fat meal with and without red wine. He found post-meal plasmaperoxide levels were much lower in those who drank wine. (Ursini F, etal. Post-prandial plasma peroxides: a possible link between diet andatherosclerosis. Free Rad Biol Med 1998; 25:250-2.)

A steady stream of animal and in vitro studies supplemented byepidemiological evidence and a smattering of preliminary human studiesreveal numerous health benefits associated with these compounds. Chiefamong the benefits is antioxidant protection against heart disease andcancer.

Proanthocyanidins—more technically oligomeric proanthocyanidins and,hence, the OPC moniker—are a class of flavonoids. Formerly called“condensed tannins,” all proanthocyanidins are chemically similar, theonly differences being slight changes in shape and attachments of theirpolyphenol rings. In nature, a jumble of different proanthocyanidins isalways found together, ranging from individual units to complexmolecules of many linked units (oligomers).

Proanthocyanidins are a highly specialized group of bioflavonoids thathave been extensively studied since the late 1960's for their vascularwall strengthening properties and free radical scavenging activity.Proanthocyanidins are one of the most potent free radical scavengersknown, possessing an antioxidant effect up to 50 times more potent thenvitamin E and up to 20 times greater than vitamin C. Proanthocyanidinsalso have an affinity for cell membranes, providing nutritional supportto reduce capillary permeability and fragility. Although bioflavonoidsare widespread in nature, the powerful proanthocyanidin compound is mostabundant and available from the bark of the maritime pine and grapeseeds, or pips.

Bilberry extract contains anthocyanidins with claimed visual anddemonstrated vascular enhancing properties. Bilberry is claimed toreduce visual fatigue and improve light to dark adjustment through itsaffinity for the rhodopsin-opsin system, the pigment system whichmediates both light and dark vision and visual adaptation to dimly litspaces. However, two military studies done in Israel and the UnitedStates have failed to find any such benefit from bilberry extract. Theextract may, however promote the retina's own enzymatic antioxidantdefenses.

In the vascular system the anthocyanidin extract supports the integrityof vascular walls by increasing vitamin C levels within cells,decreasing the permeabilizing effect of certain proteolytic/lysosomalenzymes, stabilizing cell membranes, and stimulating the synthesis ofcollagen and connective ground substance tissue.

Grape pips (seeds) are a potent source of proanthocyanidins, orpycnogenols. Jacques Masquelier, Ph.D., who pioneered proanthocyanidinresearch and coined the term “pycnogenol,” used the grape seed extractin his second phase of proanthocyanidin investigations.

In vitro studies suggest OPCs also provide cancer protection. OPCs inVaccinium—family berries, including blueberry, lingonberry andcranberry, block tumor growth by preventing protein synthesis in tumorcells, which prevents them from multiplying. (Bomser J, and Madhavi D.L. In vitro anticancer activity of fruit extracts from Vacciniumspecies. Planta Med, 1996; 62:212-6.) Also in the laboratory, barleybran OPCs transformed human myeloid leukemia cells into cells that wereno longer cancerous. (Tamagawa K, and Fukushima S. Proanthocyanidinsfrom barley bran potentiate retinoic acid-induced granulocytic andsodium butyrate-induced monocytic differentiation of HL6O cells. BiosciBiotechnol Biochem, 1998; 62:1483-7.) Another in vitro study found thata patented grape seed extract killed cancer cells; inhibited growth ofhuman breast, lung, stomach and myelogenous leukemia cells by up to 73percent; and enhanced normal cell growth. (Ye, X. and Krohn. R. L. Thecytotoxic effects of a novel 1H636 grape seed proanthocyanidin extracton cultured human cancer cells. Mol Cell Biochem, 1999; 196:99-108.)

Proanthocyanidins may protect the body from a number of potentiallytoxic agents. Acetaminophen, the active ingredient in Tylenol™, is apotent liver toxin, annually causing 75,000 cases of poisoning requiringhospitalization in the United States. Animal experiments have shown thata week of pretreatment with 100 mg/kg of a patented grape seed extractprevented liver damage from acetaminophen. Organ damage was assessed bystudying liver cells for damage and also by monitoring the animal'shealth. (Ray S D, et al., A novel proanthocyanidin 1H636 grape seedextract increases in vivo bcl-XI expression and preventsacetaminophen-induced programmed and unprogrammed cell death in mouseliver. Arch Biochem Biophys., 1999; 369(1):42-58.)

Proanthocyanidins may do even more than prevent disease; they may helpslow the aging process and reduce visible signs of aging. Oxidationdamage causes most visible signs of aging in our skin. By preventingthis damage, skin will stay younger looking. One way to achieve this isto reduce the damaging effects of ultraviolet (UV) light. Sunscreenproducts have incorporated a variety of antioxidants with the intentthat they will prevent sun injury to the skin. In one study, grape seedOPCs exerted a solo antioxidant effect at a level of potency on a parwith vitamin E—protecting different polyunsaturated fatty acids from UVlight-induced lipid peroxidation. (Carini M., et al. The protection ofpolyunsaturated fatty acids in micellar systems against UVB-inducedphoto-oxidation by procyanidins from Vitis vinifera L., and theprotective synergy with vitamin E. Intl J Cosmetic Sci., 1998;20:203-15.) In this same study, the grape OPCs synergisticallyinteracted with vitamin E, recycling the inactivated form of the vitamininto the active form and thus acting as a virtual vitamin E extender.

Part of the aging process is the degradation of skin by the enzymeelastase, which is released with the inflammatory response. OPCsspecifically block elastase, thus maintaining the integrity of elastin.(Meunier M T, and Villie F. The interaction of Cupressus sempervirens L.proanthocyanidolic oligomers with elastase and elastins. J Pharm Beig.,1994; 49: 453-61.)

OPCs may even help growth of a thicker head of hair, if the results ofanimal experiments apply to humans. Japanese researchers shaved mice andfound that 40 percent of their hair grew back naturally. When a 1percent solution of any of three proanthocyanidins was applied to theskin, however, between 70 and 80 percent of the hair grew back. Testtube studies confirm that OPCs actually stimulate the hair keratinocytesto produce three times more hair than the controls. (Takahashi T, et al.Procyanidin oligomers selectively and intensively promote proliferationof mouse hair epithelial cells in vitro and activate hair folliclegrowth in vivo. J Invest Dermatol., 1999; 112:310-6.)

B. Phenolic Compounds: Luteolin-4′-glucoside

Inhibits proinflammatory cytokine production in macrophages. Ananti-cancer flavonoid: poisons eukaryotic DNA topoisomerase I.

II. Measurement of Anthocyanins in Freeze-Dried Jucara Powder

The anthocyanin profile of freeze-dried Jucara powder was measured byLC/MS/MS and is shown in FIG. 2. The LC/MS/MS results for peaks shown inFIG. 2 are summarized below in Table 7.

Anthocyanin and OPC analysis (phenolic compounds) was performed asdetailed below. Briefly, powdered sample was simultaneouslydifferentially extracted into water and ethyl acetate. Each layer wascollected and filtered void of solids. Intact anthocyanins were analyzedfrom the water layer by HPLC on a column of C-18 Zorbax 5 μm 150×4.6 mmusing a gradient mobile phase (1 ml/min. flow) consisting of A (0.5%phosphoric acid) B (water:acetonitrile:acetic acid:phosphoricacid—50:48.5:1:0.5) and the following program—initial 100% A, 20 min 80%A, 30 min 40% A, 36 min 80% A. Identification/quantification performedby external standards. Oligomeric proanthocyanins were analyzed from theethyl acetate layer following evaporational drying, and reconstitutionin anhydrous methanol. Chromatography performed on a Phenyl-hexyl Luna 3μm 250×3.5 mm using a gradient mobile phase (1 ml/min flow) consistingof A (water:acetonitrile:acetic acid—89:9:2) B (wateracetonitrile—20:80) and the following program—initial 100% A, 25 min 60%A, 32 min 100% B, 40 min 100% A. Identification/quantification performedby first principles based on extinction coefficients of parentepicatachin and catachin ring structures.

TABLE 7 Peak Retention Time Molecular Ion Product Ion I 27.29 449 287 II28.78 595 449, 287

The structures of anthocyanins from freeze-dried Jucara powder are shownin FIG. 3.

III. Measurement of Anthocyanins in Freeze-Dried Açai Powder

The anthocyanin profile of freeze-dried Açai powder was measured byLC/MS/MS and is shown in FIG. 4. The LC/MS/MS results for peaks shown inFIG. 4 are summarized below in Table 8.

TABLE 8 Peak Retention Time Molecular Ion Product Ion I 27.42 449 287 II29.19 595 449, 287

Analysis performed as detailed above.

The structures of anthocyanins from freeze-dried Açai powder are shownin FIG. 5.

IV. Content of Anthocyanins for Freeze-Dried Jucara Powder andFreeze-Dried Açai Powder

The contents of anthocyanins measured in freeze-dried Jucara andfreeze-dried Açai powder is summarized below in Table 9.

TABLE 9 Contents of Anthocyanins Anthocyanin (mg/g) Jucara AçaiCyanidin-3-glucoside 3.43 1.77 Cyanidin-3-glucoside-coumarate 17.56 3.93Total: 20.99 5.7

The structures of the individual phenolic compounds present infreeze-dried Jucara powder are shown in FIG. 7. Significant amounts ofphenolic compounds in freeze-dried Açai powder were not detected.Analysis was performed as detailed above.

VI. Measurement of Proanthocyanidins in Freeze-Dried Açai Powder andFreeze-Dried Jucara Powder

The proanthocyanidin profile of freeze-dried Açai powder andfreeze-dried Jucara powder was chromatographically determined and isshown in FIG. 8. As detailed in FIG. 8, the profile of proanthocyanins.B1 are epicatechin and catechin. Peaks B2 through B8 stand for the Btype procyanidin from dimers to octamers. A2 are dimers with one A typeinter-flavan linkage as reflected by the mass spectra. The results forpeaks shown in FIG. 8 are summarized below in Table 11.

TABLE 11 Content of proanthocyanidins in freeze-dried samplesProanthocyanidins (mg/g, mean ± SD, n = 3) Jucara Açai Monomers 0.350.21 Dimers 0.52 0.30 Trimers 0.29 0.25 Tetramers 0.87 0.32 Pentamers0.50 0.31 Hexamers 1.03 0.52 Heptamers 0.60 0.32 Octamers 0.72 0.39Nonamers 1.40 0.64 Decamers 0.55 0.34 Polymers 18.53 9.28 Total: 25.3812.89

Jucara contains very high level of proanthocyanidins, as well as, highantioxidant activities against hydroxyl radical and peroxynitrite. FIG.9 shows representative structures of proanthocyanidins detected infreeze-dried Açai powder and freeze-dried Jucara powder. Analysis wasperformed as detailed above.

Example 8 Composition Analysis of Anthocyanin Content of Freeze-DriedAçai Berry Powder

Composition analysis of the anthocyanin content of freeze-dried Açai FDberry powder (lot#MAL001) was performed by IBC Labs (IntegratedBiomolecule Corporation, Tucson, Ariz.). The results are detailed belowin Table 12.

TABLE 12 Analyte Result Unit Anthocyanins Cyanidin-3-glucoside 1.566mg/g Cyanadin-3-glucoside-6′ coumarate 4.121 mg/g Total authocyanins:5.687 mg/g OPC Degree of oligomerization (includes linear/branched) One0.5944 mg/g Two 0.4082 mg/g Three 0.7988 mg/g Four 0.8124 mg/g Five0.6821 mg/g Six 0.5223 mg/g Seven 0.4046 mg/g Eight 0.3121 mg/g Nine andabove 7.2067 mg/g Total oligomeric proanthocyanins: 11.7416 mg/g

Anthocyanin and OPC analysis was performed according to methodologyemployed by Brunswick Laboratories. Briefly, powdered sample wassimultaneously differentially extracted into water and ethyl acetate.Each layer was collected and filtered void of solids. Intactanthocyanins were analyzed from the water layer by HPLC on a column ofC-18 Zorbax 5 μm 150×4.6 mm using a gradient mobile phase (1 ml min.flow) consisting of A (0.5% phosphoric acid) B (wateracetonitrile:acetic acid:phosphoric acid—50:48.5:1:0.5) and thefollowing program-initial 100% A, 20 min 80% A, 30 min 40% A, 36 min 80%A. Identification/quantification performed by external standards.Oligomeric proanthocyanins were analyzed from the ethyl acetate layerfollowing evaporational drying, and reconstitution in anhydrousmethanol. Chromatography performed on a Phenyl-hexyl Luna 3 μm 250×3.5mm using a gradient mobile phase (1 ml/min flow) consisting of A(water:acetonitrile:acetic acid—89:9:2) B (water:acetonitrile—20:80) andthe following program—initial 100% A, 25 min 60% A, 32 min 100% B, 40min 100% A. Identification/quantification performed by first principlesbased on extinction coefficients of parent epicatachin and catachin ringstructures.

Example 9 Fatty Acid Analysis of Freeze-Dried Açai

Fatty acid analysis for freeze-dried Açai pulp was performed bySilliker, Inc. Illinois Laboratory (Chicago Heights, Ill.; laboratory IDNo. 170547512). The results are detailed below in Table 13, Table 14 andTable 15.

TABLE 13 SATURATED FATTY ACID FORMULA % Butyric  4:0 <0.1 Captoic  6:0<0.1 Caprylic  8:0 <0.1 Capric 10.0 <0.1 Undecanoic 11:0 <0.1 Lauric12:0 0.1 Tridecanoic 13:0 <0.1 Myristic 14:0 0.2 Pentadecanoic 15:0 <0.1Palmitic 16.0 24.1 Marganic 17:0 0.1 Stearic 18:0 1.6 Nonadecanoic 19:0<0.1 Eicosanoic 20:0 <0.1 Behenic 22:0 <0.1 Tricosanoic 23.0 <0.1Lignoceric 24:0 <0.1

TABLE 14 MONOUN- POLYUN- SATURATED SATURATED FOR- FATTY ACID FORMULA %FATTY ACID MULA % Tridecenoic 13:1 <0.1 Linoleic 18:2 12.5 Myristoleic14:1 <0.1 Linolenic 18:3 0.8 Pentadecenoic 15:1 <0.1 Gamma Linolenic18:3G <0.1 Palmitoleic 16:1 4.3 Elcosadienoic 20:2 <0.1 Margarolleic17:1 0.1 Elcosatrienoic 20:3 <0.1 Oleic 18:1C 56.2 Homogamma 20:3G <0.1Linolenic Elaidic 18:1T <0.1 Arachidonic 20:4 <0.1 Gadoleic 20:1 <0.1Elcosapentaenoic 20:5 <0.1 Erucic 22:1 <0.1 Docosadienoic 22:2 <0.1Nervonic 24:1 <0.1 Docosahexaenoic 22:6 <0.1

TABLE 15 Total Monounsaturated Fatty Acid 60.60 61% monounsaturatedTotal Saturated Fatty Acid 26.10 26% saturated Total PolyunsaturatedFatty Acid 13.30 13% polyunsaturated

Unless otherwise specified, all methods were performed as described inthe Official Methods of Analysis of AOAC International, 17th Edition,2000 (hereinafter, AOAC). The fatty acid profile of test sample wasmeasured using AOAC method reference #969.33.

Example 10 Fatty Acid Analysis of Freeze-Dried Jucara Fruit

Fatty acid analysis of freeze-dried Jucara fruit was performed bySilliker, Inc. Illinois Laboratory (Chicago Heights, Ill.; laboratory IDNo. 171378575). The results are detailed below in Table 16, Table 17 andTable 18.

TABLE 16 SATURATED FATTY ACID FORMULA % Butyric  4:0 <0.1 Captoic  6:0<0.1 Caprylic  8:0 <0.1 Capric 10.0 <0.1 Undecanoic 11:0 <0.1 Lauric12:0 0.1 Tridecanoic 13:0 <0.1 Myristic 14:0 0.1 Pentadecanoic 15:0 <0.1Palmitic 16.0 24.1 Marganic 17:0 0.1 Stearic 18:0 1.7 Nonadecanoic 19:0<0.1 Eicosanoic 20:0 0.2 Behenic 22:0 <0.1 Tricosanoic 23.0 <0.1Lignoceric 24:0 <0.1

TABLE 17 MONOUN- POLYUN- SATURATED SATURATED FOR- FATTY ACID FORMULA %FATTY ACID MULA % Tridecenoic 13:1 <0.1 Linoleic 18:2 10.0 Myristoleic14:1 <0.1 Linolenic 18:3 1.1 Pentadecenoic 15:1 <0.1 Gamma Linolenic18:3G <0.1 Palmitoleic 16:1 4.3 Eicosadienoic 20:2 <0.1 Margarolleic17:1 0.1 Eicosatrienoic 20:3 <0.1 Oleic 18:1C 56.2 Homogamma 20:3G <0.1Linolenic Elaidic 18:1T <0.1 Arachidonic 20:4 <0.1 Gadoleic 20:1 <0.1Eicosapentaenoic 20:5 <0.1 Erucic 22:1 <0.1 Docosadienoic 22:2 <0.1Nervonic 24:1 <0.1 Docosahexaenoic 22:6 <0.1

TABLE 18 Total Polyunsaturated Fatty Acid 11.10 Total MonounsaturatedFatty Acid 60.20 Total Saturated Fatty Acid 28.70

Unless otherwise specified, all methods were performed as described inthe Official Methods of Analysis of AOAC International, 17th Edition,2000 (hereinafter, AOAC). The fatty acid profile of test sample wasmeasured using AOAC method reference #969.33.

Example 11 Amino Acid Analysis of Freeze-Dried Açai

I. Amino Acid Analysis by Ion-Exchange Chromatography with Post-ColumnDerivation

Amino acid analysis was performed by the general procedures describedbelow.

A. Principle

This method quantitatively determines amino acid content by hydrolysiswith 6N hydrochloric acid followed by ion-exchange chromatography.O-phthaldehyde is used for post-column derivation and subsequentfluorometric detection.

B. Scope

This procedure is applicable to ingredients, mixed feeds proteincontaining substance.

C. Critical Points

Avoid excess evaporation time while drying samples. The loss of someamino acids may take place.

D. Reagents and Chemicals and Protocol

1. Water, HPLC grade, EM Science EM WX0004-1 or in-house waterpurification system.

2. O-phthaldehyde, reagent grade, Anresco 0317.

3. Amino Acid standard solution, 2.5 pmoles/mL, Sigma A9531.

4. Methanol, HPLC grade, chempure 831-295 or equivalent.

5. Brij 3 solution, 30% (w/w), Sigma 430Agr.6

6. 2-Mercaptoethanol, (2-Hydroxyethylmercaptan), Sigma M-6250.

7. L-Norleucine, Sigma N-6877.

8. Pickering buffers, pH 2.2, 3.28, and 7.40, Picketing laboratories Na220, Na 328, and Na 740.

9. Potassium hydroxide, pellets, Chempure 831-706.

10. Sodium hydroxide, pellets, Chempure 832-050.

11. Hydrochloric acid, 6 N volumetric solution, Chempure RR-155.

12. Ethylenediaminetetraacetic Acid EDTA Tetrasodium salt, hydrate,Sigma ED4SS.

13. Nitrogen source.

14. Boric acid, Chempure 830-314.

15. Norleucine Internal Standard—Weigh on an analytical balance to 0.1mg, 0.1640 g of Norleucine. Transfer to 1000 mL volumetric flask. Add250 mL HPLC water. Add 1 mL concentrated hydrochloric acid and mix. Maketo volume with HPLC water, mix and sonicate. This solution will contain1.25 p×n/mL L-Norleucine. Refrigerate to avoid bacterial growth.

16. Amino Acid Standard Solution—Warm the vial of amino acid standardsolution to room temperature. Pipes 5.0 mL into a 50 ml. volumetricflask. Pipet 10.0 mL of 1.25 pm/mL L-Norleucine internal standard intothe same 50 mL flask. Make to volume with HPLC water. Mix well andsonicate for several minutes. Transfer the standard into 4 mL Waterssample vials. Store at 0 degrees C.

17. Potassium hydroxide solution, 50%—On a top loading balance, weigh150 g of potassium hydroxide into a tared 1-liter Nalgene container.Dissolve with 150 g of deionized water, stir as necessary. Allow thesolution to cool to room temperature before usage.

18. Boric acid buffer—Weigh 122 g of boric acid into a tared 2000 mlbeaker and add 1800 ml. of HPLC water. Adjust the pH to 11.0 with 50%potassium hydroxide solution. Transfer the solution to a 4-liter glassjug and fill to volume (4 liters) with HPLC water and mix well. Thefinal solution pH should be 10.4.

19. Pickering Buffer Mobile Phase:

a. pH 3.28—This buffer may be used as is from the bottle. Filter througha 0.45 pm filter membrane and degas prior to HPLC usage by vacuum undersonication.

b. pH 7.40—This buffer may be used as is from the bottle. Filter througha 0.45 pm filter membrane and degas prior to HPLC usage by vacuum undersonication.

20. Sodium hydroxide, 0.2 N—Weigh 16 g sodium hydroxide pellets into a2-liter volumetric flask. Add approximately 1000 mL HPLC water and mixuntil the sodium hydroxide is dissolved. Weigh 0.5 g EDTA, add to thevolumetric. Make to volume with HPLC water, mix and filter through a0.45 mm filter membrane. Use plastic gallon jug as a reservoir for HPLC.Filter periodically.

21. O-phthaldehyde Weigh 1.4 g of o-pbthaldehyde (OPA) crystals into a50 mL beaker. Add 20 mL HPLC grade methanol and sonicate until thecrystals are dissolved. Add solution to a 2-liter volumetric flaskcontaining approximately 1500 ml of boric acid buffer and mix. In ahood, add 4.0 mL 2-mercaptoethanol. Fill to volume with boric acidbuffer and mix. Filter the solution through a 0.45 pm filter. Pour thefiltered solution into two 1-liter Nalgene bottles and add 3.0 mL Brj-35to each bottle. Cap the bottles with nitrogen and mix well. Refrigerateuntil needed. OPA solution is stable for approximately 1 week underthese conditions (may extend up to 2 weeks).

E. Equipment and Apparatus

1. Waters model 712 B autoinjector or equivalent.

2. Waters model 6000 pump (2), Waters 2100 or equivalent.

3. Digital Pro 380 with Waters Expert software or equivalent.

4. Kratos FS-950 fluorometric detector or equivalent.

5. Kratos URS 051 post column pump or equivalent.

6. Fiatron column heater, Eppendorf CII-30 or equivalent.

7. Fisher Isotemp Oven, model 215 P or equivalent.

8. Savant Speed Vac Concentrator, model SVC-20011.

9. Savant refrigerated condensation trap, model RT-490.

10. Savant chemical trap, model SCT-120.

11. Savant disposable cartridge for acid vapor neutralization, modelDC12OA.

12. Precision direct drive vacuum pump, model Dd-310 or equivalent.

13. Vacuum gauge, Waters Pico-Tag work station or equivalent.

14. Glass-Col small pulsing vortexer, model S8216, Glas-Col PV6.

15. Beckman pH140 Meter, Beckman 123118 or equivalent.

16. Mettler Analytical balance, model AE16O or equivalent.

17. Millipore solvent filtration apparatus, Waters 85116.

18. Interaction-Sodium loaded ion exchange column, with guard column.Interaction Chromatography AMI 1.

19. Bransonic Ultrasonic bath model 220.

20. Mettler top loading balance, model P-1000.

21. Pipeman. Gilson, 1 ml and 5 ml, Rainin P4000 and P-5000.

22. Universal lit pipes tips, 1 mL SoS mL, Rainin.

23. Plastipak syringe with Luer-Lok, 3 cc× 1/10 cc, BI) 9585 orequivalent.

24. Syringe filters, polypropylene, Teflon, 0.45 micron, Nalgene199-2045.

25. Magna Nylon 66 membrane 47 mm diameter, 0.45-micron pore size,Fisher N045P0410.

26. Repipet II Dispenser, S mL, Fisher 13-687-62A.

27. Universal fir pipes tips, 200-100C) d. VWR 53508-819.

28. Disposable culture tubes, 12×75 mm, borosilicate glass, VWR60825-550 or equivalent.

29. Sample vial assembly, 4 mL, includes taps and PFTE septa, Waters73018.

30. Low volume insert with springs, plastic, for 4 mL sample vial.Waters 72163.

31. Firestone valve, rapid purge, Ace Glass mc, 8766.

32. Culture tubes, disposable, 20×150 mm, screw cap, borosilicate glass,VWR 60826.280.

33. Screw caps for disposable culture tubes, 20 mm 0]), PTFB liner, VWR60828-570.

34. Brinkman centrifugal mill, model ZM-1 (with 0.5 ruin screen) orequivalent.

F. Sample Preparation:

The sample was ground as fine as possible while keeping moisture loss toa minimum. The sample was ground through a Brinkman Centrifugal Grindingmill model ZM-1, or equivalent, using a 0.5 mm screen to obtain a finegrind.

G. Procedure:

1. The analytical balance was calibrated and set to zero.

2. It is helpful to have knowledge of the protein content of the samplebefore weighing for amino acid analysis. With this in mind, the sampleequivalent to 20 mg protein was weighed on an analytical balance. (Referto the supplement for Free Amino Acid Determination.) For a nearly puresample, approximately 38 mg was weighed. The weight was recorded in alaboratory notebook. The sample was then quantitatively transferred to amarked 20×150-turn screw top culture tube.

3. Using the Repipet II Dispenser, 15 mL 6 N hydrochloric acid was addedto each culture tube. Because of the grind and limited amount of sampletaken, any drafts that might cause a loss of sample from the culturetube were avoided.

4. In the hood, 75 microliter of 2-mercaptoethanol was added to eachculture tube (Note 1). This allowed for better determination of theL-methionine peak.

5. Each culture tube was firestoned (Note 2), alternating betweennitrogen (10-12 psi) and vacuum at least 5 times each.

6. While the sample was under nitrogen, PTFE-faced cap were screwed on.

7. Culture tubes were placed in an oven at 110 degrees C.±2 degrees C.for 24 hours.

8. The Savant evaporation system was assembled. The power to the systemwas started at least 2 hours prior to usage, so the refrigeration unithas a sufficient amount of time to attain the final working temperatureof −92 degrees C. Oil in the vacuum pump was thoroughly degassed. Thiswas done, as needed, by opening the gas ballast valve on the pump andswitching the pump on. One hour was generally sufficient. The pump wasturned off and the gas ballast valve was closed upon completion.

9. After 24 hours, the culture tubes were removed from the oven andallowed to cool to room temperature.

10. 5 mL of HPLC grade water was added to each culture tube. The cap wasscrewed on and mixed well.

11. 5 mL of Norleucine Internal standard (1.25 pm/mL) was added to eachculture tube (Note 3). (At this point the analysis may be stopped untilthe following day if necessary. Store the culture rube at 0 degrees C.if you need to store overnight.)

12. Using a 1 mL pipetman, 2 mL of hydrolysate was transferred into amarked 12×75 mm disposable culture tube.

13. The lid to the speed vac concentrator was opened and tubescontaining the 2 mL of hydrolysate were placed into positions around therotor so that the load was well balanced.

14. The lid was closed, the vent opened and the centrifuge was started.When the rotor reached its operating rpms, the vent on the vacuum gaugewas closed and the vacuum pump was started. The evaporation process maytake place overnight, if necessary. This would be so, if many sampleswere evaporated, starting late in the day (Note 4).

15. When samples are dry (vacuum gauge reads less than 500 milliliter),the vent was slowly opened to bleed air into the chamber. The pump wasturned off and, once the chamber had been completely vented, thecentrifuge was turned off, the tubes removed.

16. 3 mL of Pickering sodium diluent 220 was added to each tube andmomentarily sonicate prior to vortexing.

17. A 0.45 pm filter was attached to a 3 mL syringe. The preparedhydrolysate was filtered into a marked 4 mL vial containing a Waters Lowvolume insert with spring, then place on 712B WISP autosampler tray.

18. HPLC Conditions:

a. All buffer and OPA solutions were degassed by sonication undervacuum. The buffer lines were placed into appropriate buffer solutionand OPA line into OPA solution. The column was equilibrated with buffers3.28 with the flow rare at 0.5 mL/minute for at least 20 minutes.

b. The sensitivity control on fluorometer was set to 450, range to 0.5,time constant to 1 second, background suppression to “to”.

c. The column heater temperature was set at 60 degrees C. and monitoredduring the run.

d. The OPA pump was started and the flow rate was set to 0.50 mL/minute(adjusting downward as necessary).

e. A standard was placed in position #1 and #2 on WISP. Multi methodand/or method table were built and 20 F1 of standard was injected.Allowed 60 minutes for run dine. Observed resulting chromatogram.Injected a third time if chromatography is not satisfactory.

f. Ran a standard after every five samples. Updated response factorswill be generated and used for subsequent injections.

g. If the peak was outside the window, the samples were reprocessed andthe retention time was adjusted in calibration table to march that ofthe sample. New corrected chromatogram were printed and stored.

h. Gradient elution using 2 buffers: Using Waters software, asatisfactory gradient was established using 2 buffers for elation ofamino acids. Pump Table 19 follows:

TABLE 19 Pump Table Standard Profile Time Flow % A % B Curve No. TotalFlow Initial 0.500 100 0 — 0.500 35.0 0.500 0 100  6 0.500 45.0 0.500 0100 11 0.500 45.5 0.500 100 0 11 0.500 Where A = Pickering Buffer Na328; B = Pickering Buffer Na 740

J. Calculations:Response factor=Amount amino acid (mg)×Area norleucine (internalstandard)

Area Amino AddThen RF×Area amino acid sample=Concentration of amino acid (mg)

Area Norleucine Internal Standard

Since the sample volume determines the final concentration,concentration of amino acid times the sample volume=final concentrationof amino acids.

K. Notes:

1. Mercaptoacetic acid may be used instead, if necessary—if usedBrij-35-dad to preserve Lrs quek.

2. The Firestone process consisted of alternately evacuating and purgingwith nitrogen the acid-sample solution in a sonic bath. This degassedthe solution and created an inert atmosphere above the acid thusminimizing oxidation of the amino acids during hydrolysis.

3. Using a 5 mL pipetman, calibrate with room temperature water to 5.000B±0.005 g.

4. With good vacuum, samples may freeze in the tubes. If so, afterapproximately 45 to 60 minutes, remove the tubes and warm hydrolysate ina beaker which contains hot water. Place tubes back into the rotor andcontinue evaporation.

L. Validation:

M. Quality Control:

1. Follow the standard quality assurance practices detailed in theQuality Assurance Manual.

2. A control standard (secondary standard) should be included in eachrun of samples. Casein is currently used as a control.

3. Results of the control standard are to be recorded in the laboratorynotebook.

4. Duplicate runs of the standard should not vary by more than 8%.

5. Notebooks are to be initialed and dated by the analyst performing thetest

6. Notebook entries are to be reviewed, understood, initialed and datedby another analyst in the department.

N. References:

1. JAOAC: Vol 65, No. 2, 1982, pp 496-497. Calculated Protein EfficiencyRation.

2. Degussa—Literature Digest for the Feedstuffs Industry—Amino AcidAnalysis. Chemie/Anwendungstechnik Hanau Stadtteil Wolfgang, Fed. Rep.of Germany.

3. The Peptides, Vol. 4, Amino Acid Analysis of Peptides, Ch 5. pp217-259, J R Benson, P. C. Louie and L A. Bradsbaw. Copyright 1981,Academic Press, Inc.

4. USDA Chemistry Laboratory Guidebook, G-41

5. IAOAC: Vol. 68, No. 5, 1985, pp 811-821. Sample Preparation forChromatography of Amino Acids: Acid Hydrolysis of Proteins.

II. Amino Acid Analysis of Freeze-Dried Açai Pulp

Amino acid analysis of freeze-dried Açai pulp was performed by Silliker,Inc. Illinois Laboratory (Chicago Heights, Ill.; laboratory ID No.170547512). The results are detailed below in Table 20.

TABLE 20 Analyte - Amino Acids Complete* Results** Aspartic Acid 0.83Threonine 0.31 Serine 0.32 Glutamic Acid 0.80 Glycine 0.39 Alanine 0.46Valine 0.51 Methionine 0.12 Isoleucine 0.38 Leucine 0.65 Tyrosine 0.29Phenylalanine 0.43 Lysine 0.66 Histidine 0.17 Arginine 0.42 Proline 0.53Hydroxyproline <0.01 Cystine 0.18 Trypotophan 0.13 *Reference Method -USDA 6.011 (1986) **Amino acid analysis data are presented as wt/wt %(g/100 g).

Example 12 Amino Acid Analysis of Freeze-Dried Jucara Fruit

Amino acid analysis of freeze-dried Jucara fruit was performed bySilliker, Inc. Illinois Laboratory (Chicago Heights, Ill.; laboratory IDNo. 171378575; as detailed in Example 11). The results are detailedbelow in Table 21.

TABLE 21 Analyte - Amino Acids Complete* Results** Aspartic Acid 0.12Threonine 0.04 Serine 0.05 Glutamic Acid 0.10 Glycine 0.04 Alanine 0.05Valine 0.05 Methionine 0.02 Isoleucine 0.03 Leucine 0.06 Tyrosine 0.02Phenylalanine 0.04 Lysine 0.05 Histidine 0.02 Arginine 0.04 Proline 0.05Hydroxyproline <0.01 Cystine 0.03 Trypotophan 0.06 *Reference Method -USDA MSS2 (1993) **Amino acid analysis data are presented as wt/wt %(g/100 g).

Example 13 Comparative Analysis of the Antioxidant Potential ofFreeze-Dried Açai and Select Vegetables by ORAC_(FL) Analysis

I. General ORAC Assay

The ORAC Assay was developed by Cao et al., and first reported in 1993:“Cao G, Alesslo H M, Cutler R G, Oxygen radical absorbance capacityassay for antioxidant:. Free Rad. Biol. Med. 1993:14:303-11’.Modifications were made to automate the analytical procedure and werereported in the literature in 1995: “Automated Assay of Oxygen RadicalAbsorbance Capacity with the COBAS FARA II Guohua Cao, Carl P. Verdon.Akin H. B. W U, Hong Wang and Ronald L. Prior, CLINICAL CHEMISTRY, Vol.41, No. 12, 1995”.

From that point forward, the Automated ORAC Assay received extensivecoverage and utilization, and as such, ORAC values have becomecommonplace in research and in the marketing of natural products.Brunswick Laboratories purchased two COBAS FARA 11 analyzers in 1997,replicated the automated method as developed by Cao, Prior, et al, andto date, has established an antioxidant database consisting of over 5000points of ORAC data for fruits, vegetables, beverages, grainsfunctional/engineered foods, extracts, and other natural productsources.

Brunswick Laboratories, working with the USDA, introduced a newfluorescence probe, fluorescein, which has been tested with severalhundred samples, in side-by-side comparison with beta-Phycoerythrin.Fluorescein, unlike beta-PE, does not interact with the tested samples,and being a synthetic compound, fluorescein has no measurablevariability from lot-to-lot. Most importantly, samples tested multipletimes under the same conditions maintain consistent and repeatableresults.

The development of the ORAC assay using fluorescein as the fluorescenceprobe has been conducted in cooperation with the developers of theoriginal automated ORAC Assay, where beta-PE was utilized as thefluorescence probe. Based on the extensively mechanistic studies, bothpatties lock to-the fluorescein based ORAC assay as being the newstandard ORAC procedure. The two ORAC assays are distinguished herein byusing the subscripts PE for phycoerythrin, and FL forfluorescein—ORAC_(PE) and ORAC_(FL).

II. Analysis of Freeze-Dried Açai Powder and Comparison with SelectVegetables

The antioxidant activity of freeze dried Açai powder (Brunswick Lab ID.02-0104; Brunswick Laboratories, Wareham, Mass.) was compared with theantioxidant activity of select vegetables as determined by ORAC_(FL)analysis technique (as detailed above) (FIG. 10). The ORAC value offreeze-dried Açai powder was measured as 442 μmole TE/g. This value wasmore than 10-fold greater than the ORAC value of purple cabbage (42μmole TE/g) (FIG. 10). The ORAC_(FL) analysis, utilizing fluorescein asthe fluorescent probe, provided a measure of the scavenging capacity ofantioxidants against the peroxyl radical, which is one of the mostcommon reactive oxygen species (ROS) found in the body. ORAC_(hydro)reflects water-soluble antioxidant capacity. Trolox, a water-solubleVitamin E analog, was used as the calibration standard and the ORACresult is expressed as micromole Trolox equivalent (TE) per gram.

Example 14 Comparative Analysis of the Antioxidant Potential ofFreeze-Dried Açai and Select Fresh Fruits by ORAC_(FL) Analysis

The antioxidant activity of freeze dried Açai powder (231003/0410-C;Brunswick Lab ID. 03-2096; Brunswick Laboratories, Wareham, Mass.) wascompared with the antioxidant activity of select fresh fruits asdetermined by ORAC_(FL) analysis technique (as detailed above) (FIG.11). As shown in FIG. 11, the ORAC value of freeze-dried Açai powder(536 μmole TE/g) was more than 2-fold greater than the ORAC values ofeither fresh Açai (185 μmole TE/g) or black raspberry (164 μmole TE/g).The ORAC_(FL) analysis, utilizing fluorescein as the fluorescent probe,provided a measure of the scavenging capacity of antioxidants againstthe peroxyl radical, which is one of the most common reactive oxygenspecies (ROS) found in the body. ORAC_(hydro) reflects water-solubleantioxidant capacity. Trolox, a water-soluble Vitamin E analog, was usedas the calibration standard and the ORAC result is expressed asmicromole Trolox equivalent (TE) per gram.

Example 15 Comparative Analysis of the Antioxidant Potential ofFreeze-Dried Açai and Select Fresh Fruits by ORAC_(FL) Analysis

The antioxidant activity of freeze dried Açai powder (Brunswick Lab ID.02-0104; Brunswick Laboratories, Wareham, Mass.) was compared with theantioxidant activity of select fresh fruits as determined by ORAC_(FL)analysis technique (as detailed above) (FIG. 12). The ORAC value offreeze-dried Açai powder was 442 μmole TE/g. This value was more than2-fold greater than the ORAC value of black raspberry (164 μmole TE/g).The ORAC_(FL) analysis, utilizing fluorescein as the fluorescent probe,provided a measure of the scavenging capacity of antioxidants againstthe peroxyl radical, which is one of the most common reactive oxygenspecies (ROS) found in the body. ORAC_(hydro) reflects water-solubleantioxidant capacity. Trolox, a water-soluble Vitamin E analog, was usedas the calibration standard and the ORAC result is expressed asmicromole Trolox equivalent (TE) per gram.

Example 16 Comparative Analysis of the Antioxidant Potential ofFreeze-Dried Açai and Select Fruits by ORAC_(FL) Analysis

The antioxidant activity of freeze dried Açai powder (Brunswick Lab ID.02-0104; Brunswick Laboratories, Wareham, Mass.) was compared with theantioxidant activity of select fruits as determined by ORAC_(FL)analysis technique (as detailed above) (FIG. 13). As shown in FIG. 13,the ORAC value of freeze-dried Açai powder was 442 μmole TE/g. The ORACvalue for freeze-dried Jucara powder was 1193 TE/g. The ORAC_(FL)analysis, utilizing fluorescein as the fluorescent probe, provided ameasure of the scavenging capacity of antioxidants against the peroxylradical, which is one of the most common reactive oxygen species (ROS)found in the body. ORAC_(hydro) reflects water-soluble antioxidantcapacity. Trolox, a water-soluble Vitamin E analog, was used as thecalibration standard and the ORAC result is expressed as micromoleTrolox equivalent (TE) per gram.

Example 17 Comparative Analysis of the Antioxidant Potential ofDehydrated Açai and Select Fresh Fruits by ORAC_(FL) Analysis

The antioxidant activity of dehydrated Açai powder (23100/0410-C;Brunswick Lab ID 03-2096; Brunswick Laboratories, Wareham, Mass.) wascompared with the antioxidant activity of select fresh fruits asdetermined by ORAC_(FL) analysis technique (as detailed above) (FIG.14). As shown in FIG. 14, the ORAC value of freeze-dried Açai powder(536 μmole TE/g) was more than 3-fold greater than the ORAC value ofblack raspberry (164 μmole TE/g). The ORAC_(FL) analysis, utilizingfluorescein as the fluorescent probe, provided a measure of thescavenging capacity of antioxidants against the peroxyl radical, whichis one of the most common reactive oxygen species (ROS) found in thebody. ORAC_(hydro) reflects water-soluble antioxidant capacity. Trolox,a water-soluble Vitamin E analog, was used as the calibration standardand the ORAC result is expressed as micromole Trolox equivalent (TE) pergram.

Example 18 Comparative Analysis of the Antioxidant Potential ofFreeze-Dried Açai and Select Fresh Vegetables by ORAC_(FL) Analysis

The antioxidant activity of freeze dried Açai powder (231003/0410-C;Brunswick Lab ID. 03-2096; Brunswick Laboratories, Wareham, Mass.) wascompared with the antioxidant activity of select fresh vegetables asdetermined by ORAC_(FL) analysis technique (as detailed above) (FIG.15). As shown in FIG. 15, the ORAC value of freeze-dried Açai powder(536 μmole TE/g) was more than 10-fold greater than the ORAC value ofpurple cabbage (42 μmole TE/g). The ORAC_(FL) analysis, utilizingfluorescein as the fluorescent probe, provided a measure of thescavenging capacity of antioxidants against the peroxyl radical, whichis one of the most common reactive oxygen species (ROS) found in thebody. ORAC_(hydro) reflects water-soluble antioxidant capacity. Trolox,a water-soluble Vitamin E analog, was used as the calibration standardand the ORAC result is expressed as micromole Trolox equivalent (TE) pergram.

Example 19 Comparative Analysis of the Antioxidant Potential of SelectFruits, Vegetables, and Nuts by ORAC Analysis

FIG. 16 shows the antioxidant activity of fruits, vegetables and nuts asdetermined by ORAC analysis technique (Brunswick Laboratories, Wareham,Mass.; as detailed above). The ORAC_(FL) analysis, utilizing fluoresceinas the fluorescent probe, provided a measure of the scavenging capacityof antioxidants against the peroxyl radical, which is one of the mostcommon reactive oxygen species (ROS) found in the body. ORAC_(hydro)reflects water-soluble antioxidant capacity. Trolox, a water-solubleVitamin E analog, was used as the calibration standard and the ORACresult is expressed as micromole Trolox equivalent (TE) per gram.

Example 20 Comparative Analysis of the Antioxidant Potential ofFreeze-Dried Açai and Select Nuts by ORAC_(FL) Analysis

The antioxidant activity of freeze dried Açai powder (Brunswick Lab ID.02-0104; Brunswick Laboratories, Wareham, Mass.) was compared with theantioxidant activity of select nuts as determined by ORAC_(FL) analysistechnique (as detailed above). The ORAC value of freeze-dried Açaipowder was 442 μmole TE/g. This value was more than 4-fold greater thanthe ORAC value of pecan (164 μmole TE/g) (FIG. 17). The ORAC_(FL)analysis, utilizing fluorescein as the fluorescent probe, provided ameasure of the scavenging capacity of antioxidants against the peroxylradical, which is one of the most common reactive oxygen species (ROS)found in the body. ORAC_(hydro) reflects water-soluble antioxidantcapacity. Trolox, a water-soluble Vitamin E analog, was used as thecalibration standard and the ORAC result is expressed as micromoleTrolox equivalent (TE) per gram.

Example 21 Comparative Analysis of the Antioxidant Potential ofDehydrated Açai and Select Dehydrated Fruits and Vegetables by ORAC_(FL)Analysis

The antioxidant activity of dehydrated Açai powder (BrunswickLaboratories, Wareham, Mass.) was compared with the antioxidant activityof select dehydrated fruits and vegetables as determined by ORAC_(FL)analysis technique (as detailed above) (FIG. 18). As shown in FIG. 18,the ORAC value of dehydrated Açai powder (536 μmole TE/g) was greaterthan the ORAC value of dehydrated black raspberry (340 μmole TE/g). TheORAC_(FL) analysis, utilizing fluorescein as the fluorescent probe,provided a measure of the scavenging capacity of antioxidants againstthe peroxyl radical, which is one of the most common reactive oxygenspecies (ROS) found in the body. ORAC_(hydro) reflects water-solubleantioxidant capacity. Trolox, a water-soluble Vitamin E analog, was usedas the calibration standard and the ORAC result is expressed asmicromole Trolox equivalent (TE) per gram.

Example 22 Comparative Analysis of the Antioxidant Potential ofDehydrated Açai and Select Fresh Vegetables by ORAC_(FL) Analysis

The antioxidant activity of dehydrated Açai powder (BrunswickLaboratories, Wareham, Mass.) was compared with the antioxidant activityof select fresh vegetables as determined by ORAC_(FL) analysis technique(as detailed above) (FIG. 19). As shown in FIG. 19, the ORAC value offreeze-dried Açai powder (536 μmole TE/g) was more than 10-fold greaterthan the ORAC value of purple cabbage (42 μmole TE/g). The ORAC_(FL)analysis, utilizing fluorescein as the fluorescent probe, provided ameasure of the scavenging capacity of antioxidants against the peroxylradical, which is one of the most common reactive oxygen species (ROS)found in the body. ORAC_(hydro) reflects water-soluble antioxidantcapacity. Trolox, a water-soluble Vitamin E analog, was used as thecalibration standard and the ORAC result is expressed as micromoleTrolox equivalent (TE) per gram.

Example 23 Comparative Analysis of the Antioxidant Potential ofDehydrated Fruits and Vegetables by ORAC_(hydro) Analysis

Table 22 summarizes the antioxidant activity of dehydrated fruits andvegetables (Brunswick Laboratories, Wareham, Mass.) as determined byORAC_(hydro) analysis technique (as detailed above). The ORAC_(FL)analysis, utilizing fluorescein as the fluorescent probe, provided ameasure of the scavenging capacity of antioxidants against the peroxylradical, which is one of the most common reactive oxygen species (ROS)found in the body. ORAC_(hydro) reflects water-soluble antioxidantcapacity. Trolox, a water-soluble Vitamin E analog, was used as thecalibration standard and the ORAC result is expressed as micromoleTrolox equivalent (TE) per gram.

TABLE 22 Fruits/Vegetables ORAC_(hydro) Scores Beets 120 Black raspberry340 Broccoli 130 Carrots 50 Cherries 100 Elderberry 240 (single sample)Green beans 70 Hawthorn 130 Red pepper 90 Red raspberry 210 Spinach 150Tomato 60 Wild blueberries 260 Wolfberry 220 (single sample) All valuesare ORAC_(hydro) per gram. All are averages of multiple samples, unlessotherwise stated.

Example 24 Comparative Analysis of the Antioxidant Potential ofDehydrated Fruits and Vegetables by ORAC_(hydro) Analysis

Table 23 summarizes the antioxidant activity of dehydrated fruits andvegetables (Brunswick Laboratories, Wareham, Mass.) as determined byORAC_(hydro) analysis technique (as detailed above). The ORAC_(FL)analysis, utilizing fluorescein as the fluorescent probe, provided ameasure of the scavenging capacity of antioxidants against the peroxylradical, which is one of the most common reactive oxygen species (ROS)found in the body. ORAC_(hydro) reflects water-soluble antioxidantcapacity. Trolox, a water-soluble Vitamin E analog, was used as thecalibration standard and the ORAC result is expressed as micromoleTrolox equivalent (TE) per gram.

TABLE 23 Fruits/Vegetables ORAC_(hydro) Scores Beets 120 Black raspberry340 Broccoli 130 Carrots  50 Cherries 100 Cranberry 125 Elderberry 240(single sample) Green Beans  70 Green pepper 160 Hawthorn 130 (singlesample) Red pepper  90 Red raspberry 210 Spinach 150 Tomato  60 Wildblueberries 260 Wolfberry 220 (single sample) All values areORAC_(hydro) per gram. All are averages of multiple samples, unlessotherwise stated.

Example 25 Comparative Analysis of the Antioxidant Potential ofFreeze-Dried Açai and Select Dehydrated Fruits and Vegetables byORAC_(FL) Analysis

The antioxidant activity of freeze dried Açai powder (231003/0410-C;Brunswick Lab ID. 03-2096; Brunswick Laboratories, Wareham, Mass.) wascompared with the antioxidant activity of select dehydrated fruits andvegetables as determined by ORAC_(FL) analysis technique (as detailedabove) (FIG. 20). As shown in FIG. 20, the ORAC value of freeze-driedAçai powder (536 μmole TE/g) was greater than the ORAC value ofdehydrated black raspberry (340 μmole TE/g). The ORAC_(FL) analysis,utilizing fluorescein as the fluorescent probe, provided a measure ofthe scavenging capacity of antioxidants against the peroxyl radical,which is one of the most common reactive oxygen species (ROS) found inthe body. ORAC_(hydro) reflects water-soluble antioxidant capacity.Trolox, a water-soluble Vitamin E analog, was used as the calibrationstandard and the ORAC result is expressed as micromole Trolox equivalent(TE) per gram.

Example 26 Analysis of the Antioxidant Potential of Freeze-Dried Açai byTrans-Reveratrol Analysis

The antioxidant activity of freeze dried Açai powder (231003/0410-C;Brunswick Lab ID. 03-2096; Brunswick Laboratories, Wareham, Mass.) wasdetermined by Trans-Resveratrol analysis (as detailed below) to be 1.1μg/g.

Samples were analyzed using an by HPLC chromatography using an HP 1100series HPLC equipped with a Phenomenex Luna Phenyl-Hexyl (250×4.6 mM)with 2 band prefilter and autosampler/injector, binary HPLC pump, columnheater, diode array detector, and fluorescence detector. The MobilePhase A2 was DI H₂O: Acetonitrile: Acetic Acid (89:9:2 v/v). The MobilePhase B2 was Acetonitrile: DI H₂O (80:20 v/v). All biological sampleswere stored in −80 degrees C. Freezer until ready to analyze. All dryand fruit samples were extracted with 20 ml of Methanol (MeOH). Afterextraction samples were sonicated for 1 hr. All samples were becentrifuged at 14000 rpm at 4 degrees C. for 5 min. Samples wereanalyzed at a flow rate of 1 ml/min with a run time of 35 min andpost-run time of 10 min. Retention Time was approximately 26 min. Thegradient was set as follows: 10 min at 0% B; 25 min at 40% B; 32 min at100% B; and 35 min at 100% B. Absorption at 280 nm was monitored.Quantification of compounds by HPLC is the process of determining theunknown concentration of a compound in a known solution. It involvesinjecting a series of known concentrations of the standard compoundsolution of Resveratrol onto the HPLC for detection. The chromatographof these known concentrations will give a series of peaks that correlateto the concentration of the compound injected.

Example 27 Analysis of Antioxidant Activities Against Hydroxyl Radicaland Peroxynitrite in Jucara and Açai Preparations

I. General

A. Peroxynitrite

Peroxynitrite is a cytotoxic product of nitric oxide (NO) andsuperoxide. Peroxynitrite is a far stronger oxidant and much more toxicthan either nitric oxide or superoxide acting separately.

A variety of pathologies are associated with the formation ofperoxynitrite, a potent oxidant formed from the reaction of NO withsuperoxide. This reaction is the fastest reaction NO is known toundergo, and transforms two relatively unreactive radicals into a morereactive oxidant, peroxynitrite. Peroxynitrite is invariably formed inlarger amounts when more NO is produced, and/or when an elevated levelof O₂ ⁻ prevails.

Peroxynitrite is a potent oxidant implicated in a number ofpathophysiological processes. Peroxynitrite freely travels acrosscellular lipid membranes. The calculated permeability coefficient forperoxynitrite compares well with water and is approximately 400 timesgreater than superoxide, hence is a significant biological effectormolecule not only because of its reactivity but also its diffusibility.(Lee, J., Marla, S. S. Peroxynitrite rapidly permeates phospholipidmembranes. Proc Natl Acad Sci., 1997.)

In this regard, pathologies such as diabetes, atherosclerosis, andischemia-reperfusion injury, are associated with oxidative stresscharacterized by an elevated level of O₂ ⁻ that can lead to increasedperoxynitrite formation. Recent evidence also suggests multiplesclerosis and Alzheimer's disease are associated with peroxynitriteformation. In addition, peroxynitrite has also been implicated duringischemia and reperfusion, and during sepsis and adult respiratorydistress syndrome. Ischemia and reperfusion are accompanied by anincrease in superoxide due to the activation of xanthine oxidase andNAPDH oxidase, respectively. Thus, peroxynitrite is likely to beimplicated in a number of pathologies in which an imbalance of NO and O₂⁻ occurs. The formation of peroxynitrite is desirable for non-specificimmunity but possibly not during signaling by NO.

Peroxynitrite is formed in biology from the reaction of nitric oxide andsuperoxide. The enzyme Superoxide Dismutase (SOD) lowers superoxide andprevents peroxynitrite formation (see my review: Pryor, W. A. andSquadrito, G. L. (1995). Am. J. Physiol. (Lung Cell. Mol. Physiol. 12)268, L699-L722). The chemistry of peroxynitrite: a product from thereaction of nitric oxide with superoxide). Peroxynitrite is a potentoxidant and itself can oxidize many biomolecules. Nevertheless, inbiological systems, it reacts mostly with carbon dioxide to formreactive intermediates, such as ONOOCO₂ ⁻, O2NOCO₂ ⁻, COO₃ ⁻, and NO₂.Of these intermediates, only COO₃ ⁻ and NO₂ participate in bimolecularreactions with biological target molecules; the CO₂ adducts ONOOCO₂ ⁻and O₂NOCO₂ ⁻ are too short lived and decompose before they can reactbimolecularly.

Oxidative stress, such as that caused by peroxynitrite is known todamage the vascular endothelium, a process that can lead toatherosclerosis (Thom, S. R. and Ischiropoulos, H. Mechanism ofoxidative stress from low levels of carbon monoxide. Health EffectsInstitute Research Report, number 80, 1997.)

B. Hydroxyl Radical

If the function of radicals is to destroy molecules and tissues, thenthe hydroxyl radical would be the radical's radical. It reacts atdiffusion rates with virtually any molecule found in its path includingmacromolecules such as DNA, membrane lipids, proteins, andcarbohydrates. In terms of DNA, the hydroxyl radical can induce strandbreaks as well as chemical changes in the deoxyribose and in the purineand pyrimidine bases.”

“Damaged proteins, many of them crucial enzymes in neurons, lose theirefficiency and cellular function wanes. Protein oxidation in manytissues, including the brain, has been proposed as an explanation forthe functional deficits associated with aging.

The hydroxyl radical is a third generation species of radical which isderived from hydrogen peroxide (H₂O₂), which, in turn, is derived fromthe superoxide radical through the action of the enzyme superoxidedismutase.

Hydrogen peroxide is reduced to hydroxyl radicals by the enzymesglutathione peroxidase and catalase in the presence of transition metalssuch iron or copper.

II. Results

The antioxidant activity of freeze-dried Açai powder and freeze-driedJucara powder (Brunswick Lab ID. Brunswick Laboratories, Wareham, Mass.)were determined by ORAC analysis technique (as detailed above) and issummarized below in Table 24.

TABLE 24 Measurement of Antioxidant Activities Against Hydroxyl Radicaland Peroxynitrite Samples HORAC NORAC Jucara 85 134 Açai 52 34

The HORAC result in Table 24 is expressed as micromole gallic acidequivalents per gram. The NORAC result in Table 24 is expressed asmicromole Trolox equivalents per gram.

Example 28 Analysis of Superoxide Dismutase-Like Activity andCyclooxygenase Inhibitory Activity of Açai and Jucara Preparations

I. Superoxide (O₂ ⁻) Scavenging Activity Assay (SOD)

A. Background

It is estimated one percent of total oxygen consumed by an adult (70 kgbody mass) is converted to superoxide anion. An adult at rest utilizes3.5 mL O₂/kg/min, which would result in 0.147 mole/day O₂ ⁻. O₂ ⁻ isbelieved to be cause of other reactive oxygen species such as hydrogenperoxide, peroxynitrite, and hydroxyl radicals (from hydrogen peroxide).Therefore, O₂ ⁻ scavenging capacity in human body is the first defenseline against oxidative stress. In fact, it is reported thatover-expression of superoxide dismutase and catalase in transgenic fliesextended life-span by as much as one-third, perhaps, due to decreasedoxidative stress reflected by lower protein carbonyl contents. (Orr andSohal, Science 263: 1128-1130, 1994. Superoxide scavenging capacity inblood is a very important parameter for one's antioxidant status. Thisassay is designed for accurately quantify this parameter in a highthroughput fashion.

B. Experimental Procedure

Instruments

Precision 2000 eight channel liquid handling system and Synergy HTmicroplate UV-VWAS and fluorescence reader both from Bio-tek Inc.(Winooski, Vt.).

Reagents

Hydroethidine was from Polysciences, Inc. (Warrington, Pa.). Xanthineoxidase (from buttermilk, Catalog number X4875), xanthine, superoxidedismutase (from bovine erythrocytes, catalog number S 2515) werepurchased from Sigma-Aldrich (St. Louis, Mo.).

i. Reagent Preparation

Buffer. The buffer consists of 75 mM phosphate buffer (pH 7.4)containing 100 μM diethylenetriamine pentaacetic acid (DTPA). To preparethe buffer, 0.0393 g of diethylenetriamine (DTPA) was weighed out and 10mLs of ORAC buffer working solution was added. This yielded 10 mLs of 10mM DTPA stock solution. Next, to 198 mLs of ORAC buffer working solutionwas added 2 mLs of DTPA stock solution. This yielded 200 mLs of 100 μMO₂ ⁻ buffer working solution with DTPA.

Xanthine oxidase. The xanthine oxidase suspension (in refrigerator) fromSigma was diluted 20 times by buffer to give a homogeneous solution.Take 19 mLs of O₂ ⁻ buffer and add 1.0 mL of Xanthine oxidasesuspension. This yielded 20 ml of Xanthine oxidase working solution,which was made fresh daily.

Xanthine solution. Xanthine (15 mg) was weighed and place in a clearglass bottle. 5 mLs of 0.1 N sodium hydroxide (0.1 N NaOH) was added andthe solution was vortexed and sonicated until the solid was dissolved.95 mLs of O₂ ⁻ buffer was added and vortexed. This yielded 100 mLs ofXanthine solution. The solution was kept at room temperature to avoidprecipitation of xanthine. The Xanthine solution was made fresh daily.

Hydroethidine (HE) Working Solution. Stock solution ofdihydroethidium—0.04 g of dihydroethidium was added to 20 mL ofacetonitrile. This yielded 20 mLs of HE stock solution (2 mg/mL), whichwas stored in small aliquot vials at −80 degrees C. Next, 0.125 mL ofdihydroethidium (HE) stock solution was added to 24.875 mLs of xanthinesolution. The solution was sonicated and heated until clear. Thisyielded 25 mLs of Hydroethidine (HE) working solution, which wasprepared fresh daily.

Superoxide Dismutase Working Solution (SOD). Thirty thousand units ofSOD (Sigma) was reconstituted in ten mL buffer solution. The solutionwas divided into small aliquots (0.4 mL per vial, stock solution) andkept at −20 degrees C. This yielded 3000 units, which was diluted to 30units for use (see below). 200 μL of SOD 3000 unit stock solution wasadded to 19.8 mLs of O₂ ⁻ buffer to yield 20 mLs of SOD 30 unit workingsolution.

Control. The stock solution was Manganese (III) 5, 10, 15, 20tetrachloride stock solution 1144 μM which was stored at −80 degrees C.To prepare the working solution, the stock solution was diluted 100-foldwith O₂ ⁻ buffer and vortexed. By taking 9.9 mLs of O₂ ⁻ buffer andadding 100 μL of Manganese stock solution, 10 mL of 11.44 μM Manganeseworking solution, which was placed in wells G1 and G12 as controls.

Assay Procedures

The assay was carried out on a Precision 2000 liquid handling systemwith a 96-well microplate using the following protocol:

In plate one (polypropylene) 200 μL of samples were added to wells B1,C1, E1, F1, and B12, C12, E12, F12.

200 μL of SOD working solution was added to D1 and D12 wells.

200 μL of O2- buffer was added to A1, H1, A12, and H12 wells.

200 μL of Manganese working solution was added to G1 and G12.

The reagents were loaded into the cups on rack B of the precision 2000as follows:

20 mLs of O2- Buffer in B1

20 mLs of HE in B2

20 mLs of Xanthine oxidase in B4

A ×2 dilution (ORAC ×2) was carried out on a Precision 2000. A dilutionwas carried out so that all the samples, standard, and blank werediluted by 2, 4, 8, 16, and 32 times.

25 μL of the solutions in each well were transferred to a reaction plate(polystyrene, 320 μL) followed by the addition of 150 μL HE workingsolution.

Incubate reading plate for 20 min at 37 degrees C.

After incubation, add Xanthine oxidase by running AAPH addition (B4)program. This allows 25 μL Xanthine oxidase working solution to be addedto all wells in plate #2.

After xanthine oxidase was added, place plate in platereader.

The plate and the fluorescence was read every minute for ten minuteswith excitation filter at 485±25 nm and emission filter at 590±30 nm thereadings were referenced to low well of D1 arbitrarily set at 5000units. Plate two layout (polystyrene) each well contains 150 μL HEworking solution, 25 μL sample, and 25 μL xanthine oxidase (added after30 min. preheat)

C. Data Processing

From the raw data, a linear curve was obtained and the slopes of thecurves were calculated by the KC-4 program used to control the platereader. The slopes were exported and further calculations were executedby Microsoft Excel software.

Simplified Chemical Kinetics

O₂ ⁻ was generated constantly by the following reaction catalyzed byxanthine oxidase. The rate of superoxide production was constant andpseudo-zero order to xanthine, which was in large excess in comparisonwith xanthine oxidase.xanthine+O₂→uric acid+O₂ ⁻  (1)

The superoxide formed was either reacted with HE or scavenged bysuperoxide dismutase.HE+O₂ ⁻→Oxidized HE  (2)2O₂ ⁻+O₂+H₂O₂  (3)O₂ ⁻+Sample+P  (4)

Assuming steady state concentration of O₂ ⁻, the fluorescence increaserates in the absence (Vo) and presence (V) of O₂ ⁻ scavenger (SOD) havethe following relationship:V _(o) /V=1+k ₃ [SOD]/(k ₂ ·[HE])  (5)

The plot of V_(o)/V vs [SOD] will give a linear curve with interceptionat (0, 1) and slope k₃/k₂[HE]. For an unknown sample the ratio betweenthe slopes of the unknown and the standard was:{k ₃ /k ₂ [HE]}/{k ₃ /k ₂ [HE]}=k ₃ /k ₂  (6)

Equation (5) would give relative SOD activity of a sample with unit ofmeasure of SOD unit equivalent per gram or per liter of the sampledepending on the concentrations used in plotting a sample's V_(o) V vsconcentration curve.

II. Cyclooxygenase Assays

A. Introduction

Inflammation is the response of our immune system to the intrusions bypathogens such as viruses and germs, as well as by chemical or physicalinsults. Painful sometimes, inflammation is normally healing response.But in some instances inflammation proceeds to a chronic state,associated with debilitating disease such as arthritis, multiplesclerosis, or even cancer. Research on experimental and system biologyhas shed light on the complex inflammation processes. One way, amongseveral others, to keep inflammation in check is to inhibit the activityof cyclooxygenase-2 (COX-2) which is directly associated withinflammation. It is also found that the non-steroid anti-inflammatorydrugs (NSAIDs) are excellent COX inhibitors. The beneficial actions ofNSAIDs can be associated with inhibition of COX-2 whereas their harmfulside effects (the most common one is gastrointestinal toxicity) areassociated with inhibition of COX-1. These synthetic COX inhibitorsinclude aspirin, ibuprofen, nap oxen, and celecoxib (Celebrex™). Moreresearch efforts have been discovering more selective and active COX-2inhibitors as new generation of NSAIDs.

Historically, herbal remedies for inflammation have been practiced forthousands of years. In fact, Celsus defined around AD 40 as ‘rubor,calor, dolor, tumor’ (redness, heat, pain and swelling) is, today, theinflammation symptoms. Only recently is the action mechanism for thebotanical extracts investigated at the molecular biology level usingCOX-1 and COX-2 inhibitory assay as a guide for isolation of effectivecomponents from herbal mixtures. This approach also permits a betterevaluation and optimization of the effectiveness of the pain-relievingand anti-inflammatory herbal supplements in the nutraceutical industry.To fulfill the industrial need for measuring COX inhibition capacity ofsamples of botanical origin, an in vitro COX-1 and COX-2inhibitor-screening assay was adopted, with modifications that improvethe efficiency and reduce cost. Described herein are the details ofCOX-1 and COX-2 inhibitory activity assay that is applicable tobotanical products.

B Assay Principle

COX-1 and COX-2 both catalyze the oxygenation of arachidonic acid toform prostaglandins (FIG. 1). The enzyme activity can be measured by theoxygen consumption rates. In fact, unit activity of enzyme is defined as“One unit of enzyme consumes one nmol of oxygen per minute at 37 degreesC. in 0.1 M tris-HCl buffer pH 8.0, containing 100 mM arachidonic acid,5 mM EDTA, 2 mM phenol, and 1 mM hematin”. The oxygen concentration ismonitored in real time by an Oxytherm (FIG. 2), an oxygen concentrationmeasurement system, purchased from Hansatech. The initial oxygenconsumption rate is obtained from the kinetic curve. In the presence ofinhibitors, the initial rate decreases. IC₅₀, the concentration at whichthe initial oxygen consumption rate decreases by 50%, is used to expressthe COX-1 and 2 inhibition activity. The selectivity is expressed as theratios of IC₅₀ for COX-1 and COX-2. Samples are normally dissolved indimethyl sulfoxide (DMSO), ethanol, or water.

C. Experimental Details

(1) Assay Conditions:

Instrument: Oxytherm

Buffer 0.1 M Tris-HCl, pH 8.0, with 5 mM EDTA, 2 mM phenol, and 1 mMheme

Temperature: 37 degrees C.

Initial [O2]: 212 mM

Enzyme volume: 5 mL (or ˜100 units)

Total volume: 0.5 mL

Sample volume: 5 mL

Substrate: 5 μL arachidonic acid (Conc. 10 mM in 0.01 M NaOH solution)

Heme: 5 mL (final conc. 1 mM)

Data recording speed: 5 readings per second

(2) Experimental Procedures:

Half mL of Tris buffer (incubated at 37 degrees C. oven) was added tothe reaction chamber followed by 5 mL 100 mM heme in DMSO. To thesolution, 5 mL COX-1 (or 10 mL COX-2) enzyme solution were added (usedas received from supplier). The mixture was incubated for one minute.Five mL sample (in DMSO or ethanol) was added and incubated for oneminute. Five mL arachidonic acid was added and the reaction ratemonitored. The initial rate was obtained from the slope of the kineticcurves.

Sample extraction and dissolution: Solid samples were extracted usingdimethyl sulfoxide (DMSO), ethanol, 50% acetone in water, or waterdepending on their solubility. Water-based liquid samples were testeddirectly or diluted with water when necessary. Oil based samples weredissolved in DMSO or ethanol for analysis.

Quality Control:

In order to ensure validity of the data and the normal performance ofthe oxytherm system, several quality control measures were applied.

(1) The known COX-1 and 2 inhibitor indomethacin was used as a qualitycontrol sample. Indomethacin has IC50 of 0.1 mM for COX-1 and 6.0 mM forCOX-2. The IC50 of the indomethacin was measured for each lot of enzyme.The properly working oxytherm system should give IC50 of indomethacinwithin 20% of the normal value for both enzymes.

(2) Each sample solution is tested in duplicate or triplet to obtain anaveraged ICW value.

(3) One blank (100% activity) was run in between every five samplesolutions to further ensure the reproducibility.

IC₅₀: The concentration of a sample when 50% of the enzyme activity isinhibited. Lower IC₅₀ means higher activity. IC₅₀ Ratio: This numberindicates the selectivity of the sample in inhibition of COX enzymes.When the ratio is one, there is no selectivity. If the ratio is smallerthan one, the sample inhibits COX-1 better than COX-2. If the ratio islarger than one, the sample inhibits COX-2 better. Standard deviation isabout 20%.

D. References:

1. Nathan, Nature 2002, 420: 846-52.

2. Tracey, Nature 2002, 420: 853-59.

3. Couzer and Marnett, Chemical Rev. 2003, ASAP.

4. Wu et al., J. Agri. Food Chem., 2002, 50: 701-05.

5. Smith and Marnett, Biochem, Biophys. Acta 1991, 1083, 1-17.

6. Johnson et al., Arch. Biochem. Biophys. 1995, 324: 26-34.

7. Kulmacz and Lands W. E. M. Requirements for hydroperoxide by thecyclooxygenase and peroxidase activities of prostaglandin H synthase.Prostaglandins 1983, 25, 531-40.

III. The Superoxide Anion Scavenging Potential of Açai and Freeze-DriedJucara Powders

The superoxide anion scavenging potential of freeze-dried Açai powderand freeze-dried Jucara powder were measured as detailed above(Brunswick Lab ID. Brunswick Laboratories, Wareham, Mass.). The moststudied superoxide dismutase (SOD) from a natural source is wheat sproutSOD. The SOD activity for wheat sprout is 160 to 500 unit per grambasis. By comparison, the freeze-dried Açai and freeze-dried Jucarapowders were substantially high in superoxide scavenging capability assummarized below in Table 25.

TABLE 25 Sample SOD (unit/g)* COX Inhibition (mg/g)** Açai 1,614 19Jucara 6,657 60 *Result is expressed as SOD unit equivalent per gram**Result is expressed as Aspirin mg equivalent per gram

Cyclooxygenase (COX) activity (COX Type 1, i.e., COX I; and COX Type 2,i.e., COX II) was measured in the presence and absence of freeze-driedAçai powder and freeze-dried Jucara powder (Brunswick Lab ID. BrunswickLaboratories, Wareham, Mass.) as detailed above. As summarized in Table25 (above) and Table 26 (below), freeze-dried Açai powder andfreeze-dried Jucara powder inhibited COX enzyme. As shown in Table 26,freeze-dried Açai powder and freeze-dried Jucara powder inhibited boththe COX I and COX II isozymes. Freeze-dried Açai powder and freeze-driedJucara powder, therefore, are effective in the prevention and treatmentof inflammatory diseases associated with COX I and COX II activity,e.g., arthritis.

TABLE 26 COX I COX II Sample (mg/mL) (mg/mL) Açai 6.96 12.50 Jucara 2.2010.92 *Results are expressed as IC₅₀ (50% Enzyme Activity InhibitionConcentration)

Example 29 Comparative Analysis of the Antioxidant Potential of Fruitsand Vegetables by ORAC_(HO) Analysis

FIG. 21 shows the antioxidant activity of fruits and vegetables asdetermined by ORAC analysis technique (Brunswick Laboratories, Wareham,Mass.; as detailed above). The ORAC_(FL) analysis, utilizing fluoresceinas the fluorescent probe, provided a measure of the scavenging capacityof antioxidants against the peroxyl radical, which is one of the mostcommon reactive oxygen species (ROS) found in the body. ORAC_(hydro)reflects water-soluble antioxidant capacity. Trolox, a water-solubleVitamin E analog, was used as the calibration standard and the ORACresult is expressed as micromole Trolox equivalent (TE) per gram. TheHORAC results in FIG. 21 are expressed as micromole gallic acidequivalents per gram.

Example 30 Preparation of Açai Juice

FIG. 22 is a flow chart detailing Açai juice preparation, includingwashing of the fruits and their pasteurization. A better conservation ofthe fruit and of the juice will allow consumption of the food whilepreserving its nutritional value and will ease the organization of itscommercialization, between harvests. The preparation and processingsteps of the Açai juice are shown in FIG. 22.

The hulling of the fruit can be done in several different ways and thesoftening conditions change from one producer to the other.

I. Optimization of the Pulp Extraction Process—Softening and MechanicalHulling

Fruit hulling can be performed in several ways, each producer has itsown way for processing the Açai (softening time and temperature, amountof water per kg of fruits, time that the fruit stays in the machine). Itwas necessary to optimize and systematize the pulp extraction process inorder to maintain reproducibility among our experiences and to assure agood output.

The Açai fruit hulling takes place, most of the time, in a mechanicalhulling with vertical axis, specifically elaborated and used for theAçai fruit. A picture of a representative mechanical hulling machine isshown in FIG. 23. It was designed for processing 2 kg of Açai each time,in order to minimize the amount of fruits in each processing.

The time and temperature of water for soaking the fruit varies from onefair-trader to the other. The fruit can be soaked in running water atroom temperature for some hours before beating or it can be softened inwarm water for a short time (10-20 minutes).

In the laboratory, several softening times (0, 5, 10, 15, 20, 30minutes) and soaking water temperatures were tested (30, 40, 45, 50 and60 degrees C.). Although the results suggested that there were nosignificant differences between the different conditions of softening, agreater output was observed when the Açai was softened in water at 45degrees C. for 20 minutes.

The hulling total time, as well as the amount of water and the way itwas used during this time, constitute very important variables to theoutput and density of the juice. Five total times of hulling were tested(altering from 2:30 to 5:00) and for each one, several hulling timeswere tested before putting the first dose of water, and several ways forputting the water inside the bulling machine (the time for the drippingof the juice at the end of the huffing was set to 45 seconds). The totalamount of water was 1 liter per 2 Kg of fruits (in order to get an idealjuice; not very strong, not very weak).

From these studies, it was noticed that the total time for hulling andthe way of putting water inside the hulling machine have a verysignificant effect on the outputs in dry substances. From thoseexperiences, the following protocol for the preparation of the juice wasset:

1. Weigh 2 kg of raw material (Açai).

2. Prepare 5 containers with 200 mL of water each.

3. Place the Açai in the machine, turning it on at time 0.

4. After one minute of hulling, put the first 200 mL water container atonce.

5. After 1 min 30 s; 2 min; 2 min 30 s; and 3 min, add one of each ofthe remaining four containers.

6. Leave it dripping for 45 seconds up to 3 min 45 sec.

The recycling impact of part of the juice was analyzed. Although thistechnique for preparing the juice is very popular, no differences in theoutputs of dry substances were noticed.

II. Evolution of the Microbiological Characteristics of Açai Fruit afterits Harvest

When Açai characteristics and qualities are compared at harvest and atmiddle harvest, significant changes were noticed in the organolepticqualities and in the numbers of the scientific indicators(microbiological, thy weight, color). For example, the Açai of theharvest sold in Beim, is cheap and abundant. It has good quality,because as it comes from places near Beim, and it doesn't suffer changesduring transportation. On the other hand, between harvests, Açai isproduced at lower quantities and its organoleptic characteristics areinferior to those of the Açai from harvest time and it is moreexpensive. The Açai produced between harvests comes from more distantplaces (Maranho, Maraj island), and undergoes a long trip beforereaching Beim's harbor. Time between harvest and sale/consumption offruit is very important (48 hours is pretty much the maximum time thefruit lasts after harvest at room temperature).

An increase in microbial load was noticed in the Açai purchased betweenharvests when compared to that from harvest time. During harvest time,the average load of microorganism per gram in fresh Açai was 10⁶microorganisms per gram of dry pulp—what accounts for 10 ml of juice.Between harvests, the average load of microorganism per gram increasedto 10⁹, indicating a microbial load 1000 times higher). Accordingly, itwas necessary to determine if the contamination increase was causedmainly because of low quality of the palms of those regions, poortransport conditions, or because of the increase in the time afterharvest, leading to natural growth of the microorganisms already presentat the fruit surface. Influence of time spent between harvest andprocessing was studied and then related to the increase in the microbialload.

Microbial load was measured at several time intervals during a 30-hourperiod in fresh fruits (taken 10 hours after harvest). Results indicatethat there was a measurable and regular increase in the originalmicrobial load of the fruit. The microbial load is about 10⁵-10⁶microorganism (bacteria, mold and leavens) per gram of dry pulp rightafter harvest and after 40 hours reaches a maximum value, a littlesuperior to 10⁹ microorganism per gram of dry pulp. Since the microbialload observed 40 hours after harvest was very similar to that of themiddle harvest, it is possible to conclude that the microbial loadvariation in the harvest time and out of harvest is mainly caused bynatural increase of microorganism on the surface of the fruit ratherthan the low quality of the palms in those regions.

Therefore, Açai should be used right after its harvest, before thesignificant increase of microbial load, to avoid an alteration to theproduct quality (not only a natural change caused by the microorganismincrease but also a change caused by necessity of using thermal radicaltreatment in order to preserve the product). However, methods arereducing the microbial load were assessed below.

III. Cleaning

The efficiency of cleaning methods on the decrease of microbial load ofjuice was studied. The studies were made using the middle harvest Açai,which means Açai that has an important level of contamination. Thecleaning of the fruit with hygienic water at 0.1% (v/v) concentration,before processing, allowed the microbial load to be reduced 2 to 400times (concerning the Açai cleaned with portable water without additionof chemical substances).

IV. Washing

The washing process is considered the primary step in the processing ofthe Açai juice, because it reduces the microbial load before processingthe Açai, without altering its texture significantly. In the case of theAçai fruit, washing prior to processing steps has been described ashelping to preserve the quality and integrity of the juice and,therefore, preserve the organoleptic, texture and nutritional qualitiesof the Açai juice. (Tournas, 1994).

Washing consists of placing the fruits in hot or boiling water orsteaming for some time before processing (Cruess, 1995). The choice oftreatment, aimed at decreasing the contaminating agents present on thesurface of the fruit, is explained by the physical structure of thefruit. The fruit that has only one small layer of superficial pulp, ashort contact time between the pulp and the hot water or steam leads toa positive efficiency of the treatment at not very high temperature orlong time.

Studies have been conducted not only with Açai from harvest time butalso Açai from middle harvest, trying at several different temperatures(from 75 degrees C. to 100 degrees C.) and several washing times (from 5sec to 10 min). (Rogez et al., 1996). The treatments had a significantimpact over the reduction of the microbial load (bacteria, mold, andleaven). However, the washing conditions were not effective for theinactivation of peroxidases, as these enzymes are more thermallyresistant. Only the higher temperatures for the longer periods of washtimes were able to partially reduce the activity of peroxidases (up to20%). But harsher treatments (i.e., temperatures over 80 degrees C. withtimes longer than 10 seconds) caused a separation of the fattysubstances of the juice (yellowish oil) seen on the surface of thejuice. This texture alteration reduced the acceptability of the productby consumer, because of its appearance.

As the losses in the organoleptic characteristics are much higher withmore radical washing, without being able to further reduce the microbialload, the temperature of 80 degrees C. and the time of 10 seconds wereselected as the better washing conditions for the Açai fruit.

Example 31 Methods of Açai Beverage Preparation and Standards forPreparation

I. Mixing Instructions

Açai 14:1 dehydrate requires 13 parts water/liquid to 1 part dehydrateby weight. The variety of possible beverages using Açai is almostunlimited. Three examples are provided below:

Mixture 1: Twenty-five grams of Açai powder was added to 325 ml coldwater. The mixture was blended at medium speed for at least 30 secondsto hydrate the powder adequately. If the mix can stand for a minute orso it will improves the texture.

Mixture 2: Twenty-five grams of Açai powder was added to 200 ml waterand 125 grams of ice; blend 30 seconds. The mixture was allowed to standone minute.

Mixture 3: Using 125 ml of milk or cream instead of ice makes adelicious smoothie.

Because Açai is low in sugar and vitamin C there is very little toprevent oxidation/fermentation. The presence of both sugar and Vitamin Cis recommended. The taste of pure Açai is rather bland and the color isa very dark maroon. The addition of 1-2 tablespoons of sugar or othersweetener compliments the flavor very nicely. The color can be maderedder through addition of Vitamin C (an acid). The addition of red foodcolor will also create a more appetizing or appealing appearance.Furthermore, the addition of a banana to the mixture, as well as asprinkling of granola arid garnishment of fruit, can also providecreative alternatives for the preparation of Açai beverages.

II. Equipment:

Blender; Gram Scale; Milliliter measuring device

III. Identity and Quality Standards for Açai Pulp

1. Goal

Present regulation aims at the establishment of minimum identity andquality standards that should be fit by Açai integral pulp and Açai, tobe used as beverage. This regulation does not apply to Açai pulp for anyother use.

2. Definition

Açai integral pulp and Açai are products extracted from the eatable partof the fruit of the Açai tree (Euterpe oleracea, Mart.) after beingsoftened by adequate technological method.

3. Classification

The product will be classified accordingly to the amount of water/liquidadded to the pulp, as follows:

3.1 Açai integral pulp is the pulp extracted from Açai without theaddition of water, by mechanical methods, and without filtration. It maybe submitted to a physical conservation process.

3.2 Thick or special Açai (type A) is the pulp extracted with theaddition of water, presenting more than 14% of total solids and a verydense appearance.

3.3 Medium or regular Açai (type B) is the pulp extracted with theaddition of water, presenting more than 11% and up to 14% of totalsolids and a dense appearance.

3.4 Thin or popular Açai (type C) is the pulp extracted with theaddition of water, presenting more than 8% and up to 11% of total solidsand not a dense appearance.

4. Basic Ingredients

The Açai integral pulp and the Açai are obtained from fresh, ripe andhealthy fruits, according to specifications described above, and withoutany dust, parasites or microorganisms that can make the productinappropriate to consumption.

5. Optional Ingredients

5.1 Water

The water used to the pulp extraction must be portable.

5.2 Acidulante

In the case of pasteurized Açai maintained at room temperature, citricacid may be added according to the ‘Good Manufacture Practices’ (GMP)regulations.

6. Composition

6.1 The Açai integral pulp and the Açai must have its compositionaccording to the fruit characteristics, with no alterations, mixtureswith other species fruits or any illegal practice.

6.2 The Açai integral pulp must fit the following physical, chemical andorganoleptic characteristics:

6.2.1 Physical and Chemical

TABLE 27 Minimum Maximum Total solids (g/100 g.) 40.0 60.0 Proteins(g/100 g) 5.0 — Total lipids (g/100 gms) 20.0 — Total carbohydrates(g/100 gms) 51.0 — Obs: gms = grams of dried material (total solids)

6.2.2 Organoleptical

Physical aspect: Pasty, presenting dark points prominent from the skinthat involves the fruit.

Color: Violet purple proper for the purple Açai pulp and light green forthe green Açai pulp.

Smell: Characteristic (see below).

6.3 The Açai (special, regular or popular) must fit the followingphysical, chemical and organoleptic characteristics:

6.3.1—Physical and Chemical

TABLE 28 Minimum Maximum pH (g/100 g.) 4.00 6.20 Total acidity, incitric acid — 0.27 (popular) (g/100 g) 0.35 (regular) 0.45 (special)Total lipids 20.0 60.00 Proteins (g/100 gms) 8.0 — Total sugars (g/100gms) — 40.0 Obs: gms = grams of dried material (total solids)

6.3.2 Organoleptical

Physical aspect: The emulsion must stay stable even if heated up to 80degrees C.

Color: Violet purple proper for the purple Açai pulp and light green forthe green Açai pulp.

Smell: Characteristic (see below).

6.4 The integral Açai pulp and the Açai may contain non-edible parts ofthe fruit into the limits that doesn't change the quality aridorganoleptical characteristics of the product. The integral Açai pulpand the Açai must fit all other physical, chemical, microscopical,microbiological, and organoleptic characteristics fixed in the Identityand Quality Standards for general fruit pulp.

6.5 The maximum limit for the sum of moulds and leavens in. the Açaiintegral pulp and in Açai is 5×10³.

7 Additives

The integral Açai pulp and the Açai directed for direct consume inmaximum 1 kg. pack must be maintained through physical process,forbidden the use of chemical conservants or coloring substances, exceptthe coloring obtained from the Açai fruit.

Example 32 Preparation of Freeze-Dried Açai

A method of preparing freeze-dried Açai powder is detailed in FIG. 24.As shown, Açai fruit were harvested and the pit was removed. The pulpwas then removed and frozen. Pulp from many Açai fruit was freeze-driedto yield a freeze-dried powder. The freeze-dried Açai powder was stablecompared with unprocessed preparations of the Açai fruit pulp, whichrapidly degraded within hours, rendering them unpalatable. The additionof citric acid to the Açai fruit pulp during processing and prior tofreezing was useful in further stabilizing the fruit pulp preparation.Citric acid can be used to stabilize other fruit pulp preparations,e.g., Jucara, processed by the methods of the present disclosure.

Açai production is a particularly unforgiving sequence of events due toenzymes and a proportionally high load of fermenting agents on the fruitskin compared to the quantity of pulp removed from the fruit. For thisreason, Açai production was traditionally limited to local and immediateconsumption.

Açai frozen fruit pulp must be maintained at a temperature of −5 degreesC. or less. At higher temperatures, the enzymes and fermenting agentsbecome active and change the characteristics of the fruit pulp. Oneeffect is the creation of insoluble compounds, the grit mentioned above,which is evident with this last batch. These insolubles were encounteredin the first batches of Açai dehydrate (from two processors) and werefound to be caused by the thawing of the pulp during preparation fordehydrating. This problem was resolved by not allowing the frozen Açaifruit pulp to pre-thaw prior to dehydration via freeze-drying. That is,once the Açai fruit pulp is frozen, it cannot be allowed to thaw to atemperature greater than about −5 degrees C. prior to dehydration byfreeze-drying. Açai fruit pulp prepared without pre-thawing beforedehydrating yielded a granular, freeze-dried Açai powder thatre-hydrated very successfully and retained quality color, texture andflavor. Therefore, the present disclosure provides for a method ofpreparing a fruit-based dietary supplement wherein the fruit pulp isprepared by a method wherein once the pulp is isolated and frozen it isnot allowed to pre-thaw prior to dehydration. This method is useful inpreparing freeze-dried fruit powders from many fruits, e.g., but notlimited to, Açai fruit and Jucara fruit, which can be re-hydrated andretain quality color, texture, and taste.

The granular, freeze-dried fruit powder was stored light protected in aplastic-lined foil bag until use.

Example 33 Challenge Testing of Freeze-Dried Açai Preparation forStability to Selected Microbes

I. Objective

The objective of this study was to conduct a preliminary challenge testto assess the microbiological stability of a product when challengedwith one strain each of yeast, mold, lactic acid bacteria, Salmonella,and Staphylococcus aureus. (Silliker Laboratories Research Center, SouthHolland, Ill.).

II. Applications

This study offers a screening of a product for potential spoilageorganisms and two pathogens. It is appropriate to gather initial dataabout a product and/or to compare a number of product formulationsduring development.

III. Limitations

With only one strain of each challenge organism, there is a chance thatthe product will be resistant to growth by that strain but susceptibleto other strains. In the challenge organisms grow in the controlproduct, it will be not be determined until the end of the study. Thisstudy is limited in time intervals, storage temperatures, and the scopeof the report. The study does not predict the results beyond four weeks.

IV. Material and Methods

A. Test Product

A 3.5-kilogram resealable foil bag of product labeled “Açai fruit-freezedried” was received from the client. Product was stored at ambienttemperature until initiation of the study.

B. Challenge Organisms

The product was challenged with freeze-dried strains of Aspergillusniger (mold), Zygosaccharomyces bailli (yeast); Lactobacillusfructivorans (lactic acid bacteria), Salmonella typhimurium, andStaphylococcus aureus form the Silliker Research Culture Collection(SRCC) as summarized in Table 29. The number of viable cells or sporeswas verified by plate count methods.

TABLE 29 Organism SRCC Number Aspergillus niger 1131 Zygosaccharomycesbailii 764 Lactobacilus fructivorans 464 Salmonella typhimurium 449Staphylococcus aureus 713

C. Preparation of Test Samples and Storage

The product was aseptically divided into 6 sterile containers in100-gram portions. One portion served as a negative control. The otherportions were inoculated with one of the cultures at approximately10,000 colony-forming units per gram. After inoculation, the sampleswere mixed thoroughly and stored at 75 degrees F.

D. Sample Analyses

The uninoculated control portion was analyzed for challenge organisms ondays 0 and 28. Inoculated portions were analyzed on days 0, 7, 14, 21,and 28. A single 11-gram sample was taken from each portion at eachinterval and analyzed by plate court methods for challenge organisms.

V. Results and Discussion

The microbiological stability of a food product may be determined bychallenging it with spoilage and pathogenic microorganisms. When thelevel of the challenge organisms does not increase during storage, theproduct formulation is resistant to microbial growth and is consideredmicrobiologically stable.

The results are shown in Table 30 and Table 31. As the data show, thecounts of yeast, mold, lactic acid, bacteria, Salmonella, andStaphylococcus aureus did not increase in the control or inoculatedportions of the product during storage. Thus, the Açai fruit-freezedried product was microbiologically stable for at least 28 days whenchallenged with yeast, mold, lactic acid bacteria, Salmonella, andStaphylococcus aureus and stored at 75 degrees F. As shown below, thisproduct was stable against challenge.

TABLE 30 Açai Fruit - Freeze Dried Non-inoculated Control Samples LacticAcid Yeast Mold Bacteria Salmonella Staphylococcus Interval (cfu/g)(cfu/g) (cfu/g) (cfu/g) (cfu/g) Day 0 20 <10 20 <10 <10 Day 28 10 <10 20<10 <10 cfu/g = colony forming units per gram

TABLE 31 Açai Fruit - Freeze Dried Inoculated Samples Lactic Acid YeastMold Bacteria Salmonella Staphylococcus Interval (cfu/g) (cfu/g) (cfu/g)(cfu/g) (cfu/g) Day 0 470 44,000 3,400 190 44.000 Day 7 180 50,000 2,80080 100 Day 14 10 10,000 580 170 50 Day 21 20 27,000 800 30 <10 Day 28 304,700 230 10 <10 cfu/g = colony forming units per gram

VI. Shelf-Life Studies on Freeze-Dried Açai

Shelf life studies on freeze-dried Açai preparations were conducted bySilliker Laboratories as summarized in below in Table 32.

TABLE 32 Results of shelf-life study LACTIC ACID Aerobic Plate YeastMOLD BACTERIA Month Count (CFU/g) (CFU/g) (CFU/g) (CFU/g) 0 — 20 <10 201 — 10 <10 20 2 1,500 30 40 10 3 50 <10 <10 10 4 750 <10 <10 <10 5 500<10 <10 180 6 660 <10 <10 180 7 1,200 <10 <10 <10 8 360 <10 <10 <10 9<10 <10 10 <10 10 <10 <10 10 <10 11 <10 <10 <10 <10 12 <10 <10 <10 <10CFU/g = colony forming units per gram

The taste, odor and appearance of a food (organoleptic qualities) arethe ultimate criteria used by consumers to judge a food's acceptability.These qualities begin to change as the microflora in the food-bacteria,yeast, and mold grow and metabolize available nutrients. Organolepticchanges are generally not detectable until the microbial population ishigh. The number of organisms required to cause spoilage varies with thefood item and the type(s) of microorganisms growing in it. Generally,however, the end of shelf life limits during storage. Therefore, theshelf life of the Açai Fruit-Freeze Dried product was at least 12 monthsstored at 75 degrees F.

Example 34 Acute Oral Toxicity Study of ‘Açai Fruit Pulp Freeze Dried’with 14-Day Post-Treatment Observation Period in the Rat (Limit Test)

Studies were conducted to assess the acute oral toxicity of freeze-driedAçai fruit pulp with a 14-day posttreatment observation period in therat (limit test) ((Study code: PCDL-0221; Pharmaceutical Control andDevelopment Laboratory Co. Ltd., 9. Mexikoi Street Budapest, H-1149).

I. General Information:

A. Dose

Single oral limit dose of 2,000 mg/kg body weight of ‘Açai fruitpulp—Freeze dried’ (Lot number: 22.10) was applied to rats orally bygavage. Animals were observed for lethality and toxic symptoms for 14days. Gross pathological examination was carried out on the 15th day.The body weight of the animals corresponded to their species and agethroughout the study. No death occurred after oral administration of‘Açai fruit pulp—Freeze dried’ at 2,000 mg/kg dose. No toxic clinicalsymptoms were observed. Scheduled autopsy carried out on day 15 revealedno toxic gross pathological changes. It was concluded that no adverseeffects were noted at single oral dose of 2,000 mg/kg ‘Açai fruitpulp—Freeze dried’ in male and female rats.

B. Objective

To develop data on the potential toxicological effects of single oraladministration of Açai fruit pulp—Freeze-dried in the rat. The testarticle is expected to use as dietary supplement.

C. Type of the Study

Preclinical toxicological study in compliance with the principles of theGood Laboratory Practice Regulations for Nonclinical Laboratory Studiesof the United States Food and Drug Administration and the Hungarian Act1998: XXVIII regulating animal protection. Limit test.

D. Deviations from the Study Protocol

i. Characteristics of Substance T 61 Used for ExterminationManufacturer:

Original protocol: Hoechst Veterinar GmbH

Final Report: Intervet International

Reason: The name of the manufacturer has been changed.

ii. Mortality

Original protocol: Observations are made for 4 hours following treatmentand twice daily thereafter.

Final Report Observations were made for 4 hours following treatment andtwice daily thereafter at the beginning and at the end of the workingday as well as once at weekends, until the morning of the 15th day.

Reason: Procedures have been described more precisely than originally.

iii. General State, External Appearance, Behavior, and Clinical Symptoms

Original protocol: During the post-treatment period, animals are checkeddaily twice until the morning of the 15th day.

Final Report During the post-treatment period, animals were checkeddaily twice until the morning of the 15th day except for weekends whenanimals were checked once.

Reason: Procedures have been described more precisely than originally

II. Test and Reference Articles

A. Characteristics of the Test Article

The characteristics of the test article are detailed below in Table 33.

TABLE 33 Name of the article: Açai fruit pulp - Freeze dried Botanicalname: Euterpe oleracea, Family: Palmae Plant part used: Fresh FrozenFruit Pulp Manufacturer: Greater Continents do Brasil Ltda. RuaAlabastro, 55-112, Aclimacäo 01531-010 Säo Paulo, SP Brasil Lot #: 22.10Identification number 2002/22885 in PCDL: Re-hydration: 1:13 waterResidual moisture: max. 3%, result: 1% Physical characteristics: darkpurple granular freeze dried powder with characteristic odor and flavor,hygroscopic Storage conditions: refrigerated according to USP (2-8° C.,humidity not controlled), re-sealed quickly if opened

B. Microbiological Analysis

Microbiological limit test according to c. USP was carried out by theMicrobiological Department of PCDL.

C. Characteristics of the Article Used for Suspending the Test Article

i. Methylcellulose

Methylcellulose (Bach No. 127H1066; Expiration February 2003) wascommercially obtained from Sigma and stored at room temperature prior touse.

ii. Distilled Water

Distilled water (Batch No. A0010102; Expiration March 2003) wascommercially obtained from PCDL and stored at room temperature prior touse.

iii. Characteristics of article used for overanesthesia before necropsy

T 61 (Batch No. 09W008; Expiration May 2006) containing 0.2 gembutramide, 0.005 g terracing hydrochloride, and 0.05 g mebezoniumiodide per ml was commercially obtained from Intervet International andstored at room temperature, in a safe box for poisonous drugs prior touse.

iv. Formulation of the Test Article

The necessary amount of the test article was weighed and suspended in 1%methylcellulose containing solution not earlier than 30 mm beforeadministration. The following suspension was prepared: Nominal dose 2000mg/kg: 5.0 g Açai fruit pulp ad 50 ml of 1% methylcellulose solution.Suspension was stirred during treatment with a Radelkis magnetic stirrertype OP-951.

V. Concentration Control of the Formulated Test Article

Samples of the formulated test substance were taken for check of theconcentration and homogeneity. Concentration and homogeneity check wasperformed by gravimetry. The concentration of all three samples measuredin triplicates of the upper, intermediate, and lower parts of thesuspension (homogeneity check) were within the acceptable ±10% limitsi.e., upper: +4.4±4.6%, intermediate: +4.0±4.8%, lower: +5.4±2.8%.

III. Test System

A. Animals

Sprague Dawley rat, Crl:CD BR (6-7 weeks of age at arrival) were used inthe present studies. The males had body weights that ranged from 143.9 gto 159.4 g. The females had body weights that ranged from 140.5 g to161.4 g. A pool of animals ordered: 30 (15 males, 15 females). Number ofanimals involved in the study: 20 (10 males, 10 females) Rats werecommercially obtained from Charles River Hungary Ltd. Animals were SPFat arrival and kept in a conventional environment during the study. Therat is commonly used for toxicological studies in accordance withinternational recommendations. The Sprague Dawley strain is a well-knownlaboratory model with sufficient historical data.

The animals were identified by ear numbering technique and housed incages by five of the same sex. The cages were labeled with tagsindicating the I.D. numbers of the rats, the study code, the groupidentification, route of administration, sex and the starting and endingdates of the experimental period.

The animal housing conditions are summarized below in Table 34. Theenvironmental conditions are summarized below in Table 35.

TABLE 34 Animal housing conditions Hygienic level: conventional Type ofanimal cages: type II macrolone Size of cage: H × W × D: 17.5 cm × 22.5cm × 37.5 cm Cleaning: by changing the bottom of the cages three times aweek Number of animals per cage:  5 Number of animal keeping room: 123

TABLE 35 Environmental conditions Air exchange: approximately 15times/hour Temperature: 22 ± 3° C. Relative humidity: 30-70% Lighting:artificial, 12 hour light-dark cycles.

The temperature and the relative humidity were continuously recorded.The animals were given free access to standardized rat and mouse dietVRF-1 except for the overnight fasting period prior to treatment, duringthe treatment, and for the first two hours of posttreatment observation.The composition of the diet was controlled by the Manufacturer AltrominGmbH, D-4937 Lage/Lippe Lange Str. 42. The diet was identified by thedate of manufacturing (30.09.2002), stability: 4 months. Rats had freeaccess to tap water via drinking bottles. Drinking water is checkedmonthly by the Microbiological Department of PCDL. The animals wereobserved for 5 days prior to the treatment. Only healthy animals, freefrom any clinical symptom were used in the study.

Grouping of the animals was made with a random table generated by acomputer. The animals were randomly assigned to groups on the basis oftheir body weight, so that the distribution of the body weights in theindividual groups were similar.

IV. Experimental Design

The dose levels and group division are summarized below in Table 36.

TABLE 36 Number of Dose Animals Identification # Group # Treatment mg/kgMales Females Males Females 1 Açai fruit 2,000 10 — 851-860 — pulp 2Açai fruit 2,000 — 10 — 861-870 pulp

The rationale for the dose selection is as follows. The expected humandaily dose of Açai fruit pulp is approx. 1000 mg per day whichcorresponds to 14 mg/kg body weight of an adult (70 kg) or 50 mg/kg fora 4 years old child (20 kg). The 2000 mg/kg limit dose applied in thisstudy corresponds to 140 times of the daily dose if consumed by an adultor 40 times of it if 5 g is calculated for a child's body weight.

V. Administration

Application was oral by gavage. The route of application was selected incompliance with international guidelines. The oral route is theanticipated route of human exposure to the test article. The applicationof the test article was given in a single dose. The test article wasadministered in a volume of 20 ml/kg body weight. The experimentalperiod consisted of 5 days of acclimatization, treatment's day, 14 daysposttreatment observation period including the treatment's day, and the15th day: necropsy.

VI. Observations, Examinations

A. Lethality

Observations were made for 4 hours following treatment and twice dailythereafter at the beginning and at the end of working days as well asonce at weekends until the morning of the 15th day. The time of deathshould have been recorded as accurately as possible.

B. General State, External Appearance, Behavior, and Clinical Symptoms

Careful clinical observation of the rats was carried out once before theexposure, then, after the treatment for 6 hours continuously. During thesubsequent period, animals were checked daily twice until the morning ofthe 15th day except for weekends, when animals were checked once. Signsto be observed included changes in skin, fur, eyes and visible mucousmembranes; occurrence of secretions and excretions and autonomicactivity (e.g., lacrimation, piloerection, diarrhea, pupil size, unusualrespiratory pattern). Furthermore, potential changes in gait, postureand response to handling as well as the presence of somnolence,trembling, clonic or tonic movements, stereotypes or bizarre behaviorwere recorded.

C. Body Weight

Animals were weighed at arrival in the laboratory, on the day ofrandomization, on the day of treatment, as well as on the 2nd, 8th, and15th day of the experiment prior to the necropsy.

VII. Necropsy and Histological Examination

A. Necropsy

All surviving rats on completion of the posttreatment observation periodwere exterminated under T61 overanaesthesia and autopsied. External andinternal status were carefully observed and recorded. No microscopicexamination of organs was performed.

VIII. Evaluation, Statistical Analysis

Groups of males and females were evaluated separately.

A. Parametric Values

Mean values and standard deviations were calculated of the body weights.

B. Non Parametric Values (Lethality and Clinical Symptoms)

The incidence of lethality, clinical symptoms, and gross findings weretabulated.

IX. Procedures

The experiments were performed according to the current StandardOperating Procedures of the Department of Toxicology of thePharmaceutical Control and Development Laboratory Co. Ltd.

X. Animal Protection

In the interests of animal welfare the unnecessary use of animals wasavoided. To order the mild extermination of unambiguously moribundanimals was the responsibility of the study director. The present method(limit test) uses a reduced number of experimental animals in comparisonto other known and acknowledged acute toxicity tests.

XI. Data Recording and Archivation

All original data are maintained, as dictated by the Standard OperatingProcedures, on appropriate follows:

Test Compound weighing

Animal room logbook

Body weight logbooks

Lethality and Clinical Observations Logbooks

Postmortem Records

The data obtained in the course of the study were collected in a StudyFile. The Study Protocol, all data generated during and as a result ofthe study, the documents and all information in connection with thestudy, a control sample of the test article and the Final Report will bestored at least for 15 years in the Archives of the PCDL then offered tothe Sponsor.

XII. Results

A. Lethality

The lethality observed in the 14-day post-treatment observation periodis summarized below in Table 37.

TABLE 37 Group 1 Group 2 MALES FEMALES Treatment Death/number of animalsAçai fruit pulp; 0/10 0/10 2,000 mg/kg, po.

Table 38 summarizes the individual lethality data for male test subjectsin the acute oral toxicity study of ‘Açai fruit pulp—Freeze dried with14-day post-treatment observation period in the rat (limit test).

TABLE 38 Males Group* DAYS OF OBSERVATION PERIOD Animal Day Day Day DayDay Day Day code 1* Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 1011 12 13 14 15 851 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 852 0 0 0 0 0 0 0 0 0 00 0 0 0 0 853 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 854 0 0 0 0 0 0 0 0 0 0 0 00 0 0 855 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 856 0 0 0 0 0 0 0 0 0 0 0 0 0 00 857 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 858 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0859 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 860 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0*Açai fruit pulp (2,000 mg/kg, po.); Remarks: 0 = No Lethality; *Day 1 =Treatment's day

Table 39 summarizes the individual lethality data for female testsubjects in the acute oral toxicity study of ‘Açai fruit pulp—Freezedried with 14-day post-treatment observation period in the rat (limittest).

TABLE 39 Females Group* DAYS OF OBSERVATION PERIOD Animal Day Day DayDay Day Day Day code 1* Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 910 11 12 13 14 15 861 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 862 0 0 0 0 0 0 0 00 0 0 0 0 0 0 863 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 864 0 0 0 0 0 0 0 0 0 00 0 0 0 0 865 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 866 0 0 0 0 0 0 0 0 0 0 0 00 0 0 867 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 868 0 0 0 0 0 0 0 0 0 0 0 0 0 00 869 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 870 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0*Açai fruit pulp (2,000 mg/kg, po.); Remarks: 0 = No Lethality; *Day 1 =Treatment's day

No death occurred following the single oral administration of 2,000mg/kg dose of ‘Açai fruit pulp—Freeze dried’ to rats. All males andfemales survived until the end of the 14-day observation period.

B. Clinical Symptoms

The clinical symptoms observed in the 14-day post-treatment observationperiod are summarized below in Table 40.

TABLE 40 Group 1 Group 2 MALES FEMALES Symptom/number Treatment ofanimals Açai fruit pulp; 0/10 0/10 2,000 mg/kg, po.

Table 41 summarizes the individual clinical symptoms for male testsubjects in the acute oral toxicity study of ‘Açai fruit pulp—Freezedried’ with 14-day post-treatment observation period in the rat (limittest).

TABLE 41 MALES Group* DAYS OF OBSERVATION PERIOD Animal Day Day Day DayDay Day Day code 1* Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 1011 12 13 14 15 851 SF SF SF SF SF SF SF SF SF SF SF SF SF SF SF 852 SFSF SF SF SF SF SF SF SF SF SF SF SF SF SF 853 SF SF SF SF SF SF SF SF SFSF SF SF SF SF SF 854 SF SF SF SF SF SF SF SF SF SF SF SF SF SF SF 855SF SF SF SF SF SF SF SF SF SF SF SF SF SF SF 856 SF SF SF SF SF SF SF SFSF SF SF SF SF SF SF 857 SF SF SF SF SF SF SF SF SF SF SF SF SF SF SF858 SF SF SF SF SF SF SF SF SF SF SF SF SF SF SF 859 SF SF SF SF SF SFSF SF SF SF SF SF SF SF SF 860 SF SF SF SF SF SF SF SF SF SF SF SF SF SFSF *Açai fruit pulp (2,000 mg/kg, po.); Remarks: SF = Symptom Free; *Day1 = Treatment's day

Table 42 summarizes the individual clinical symptoms for female testsubjects in the acute oral toxicity study of ‘Açai fruit pulp—Freezedried’ with 14-day posttreatment observation period in the rat (limittest).

TABLE 42 FEMALES Group* DAYS OF OBSERVATION PERIOD Animal Day Day DayDay Day Day Day code 1* Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 910 11 12 13 14 15 861 SF SF SF SF SF SF SF SF SF SF SF SF SF SF SF 862SF SF SF SF SF SF SF SF SF SF SF SF SF SF SF 863 SF SF SF SF SF SF SF SFSF SF SF SF SF SF SF 864 SF SF SF SF SF SF SF SF SF SF SF SF SF SF SF865 SF SF SF SF SF SF SF SF SF SF SF SF SF SF SF 866 SF SF SF SF SF SFSF SF SF SF SF SF SF SF SF 867 SF SF SF SF SF SF SF SF SF SF SF SF SF SFSF 868 SF SF SF SF SF SF SF SF SF SF SF SF SF SF SF 869 SF SF SF SF SFSF SF SF SF SF SF SF SF SF SF 870 SF SF SF SF SF SF SF SF SF SF SF SF SFSF SF *Açai fruit pulp (2,000 mg/kg, po.); Remarks: SF = Symptom Free;*Day 1 = Treatment's day

No toxic symptoms were observed on the day of application and during the14-day posttreatment period at any group of the treated animals.

C. Body Weights

The body weights of male test subjects observed in the 14-daypost-treatment observation period are summarized below in Table 43.

TABLE 43 MALES Body weights [g] Day of Group 1 Day of random- Treatment*arrival ization Day −1** Day 2 Day 8 Day 15 Group 10 10 10 10 10 10size: Mean: 151.3 198.8 182.2 205.4 260.5 310.0 ±S.D.: 5.52 6.60 6.437.95 16.10 14.23 *Açai fruit pulp (2,000 mg/kg, po.); **One day prior totreatment

The body weights of female test subjects observed in the 14-daypost-treatment observation period is summarized below in Table 44.

TABLE 44 FEMALES Body weights [g] Day of Group 1 Day of random-Treatment* arrival ization Day −1** Day 2 Day 8 Day 15 Group 10 10 10 1010 10 size: Mean: 148.9 177.6 161.2 180.4 202.6 232.8 ±S.D.: 7.08 6.935.71 4.25 15.80 7.66 *Açai fruit pulp (2,000 mg/kg, po.); **One dayprior to treatment

The body weight changes of male test subjects observed in the 14-daypost-treatment observation period is summarized below in Table 45.

TABLE 45 MALES Groups Body weight changes [g] Treatment* Day 1 Days 2-7Days 8-14 Group size: 10 10 10 Mean: 182.2 205.4 260.5 ±S.D.: 6.43 7.9516.10 *Açai fruit pulp (2,000 mg/kg, po.)

The body weight changes of female test subjects observed in the 14-daypost-treatment observation period is summarized below in Table 46.

TABLE 46 FEMALES Groups Body weight changes [g] Treatment* Day 1 Days2-7 Days 8-14 Group size: 10 10 10 Mean: 19.2 22.2 30.2 ±S.D.: 3.3413.00 15.73 *Açai fruit pulp (2,000 mg/kg, po.)

The individual body weights of male test subjects observed in the 14-daypost-treatment observation period is summarized below in Table 47.

TABLE 47 MALES Group Body weights [g] Animal Day of code Day of random-*Group 1: arrival ization Day −1** Day 2 Day 8 Day 15 851 159.4 207.8187.8. 214.2 287.2 314.0 852 154.4 205.9 191.1 214.7 264.6 313.1 853154.3 204.2 186.6 212.3 269.8 319.8 854 153.4 201.3 187.2 211.6 268.6330.1 855 153.7 200.8 182.7 209.2 268.2 323.2 856 147.4 199.1 177.5200.0 242.2 308.7 857 156.4 197.8 184.3 203.6 261.4 292.5 858 145.6192.1 178.1 196.1 265.2 286.4 859 143.9 191.4 175.8 197.4 232.2 316.4860 144.1 187.8 170.7 194.4 245.2 295.3 Group 10 10 10 10 10 10 size:Mean: 151.3 198.8 182.2 205.4 260.5 310.0 ±S.D.: 5.52 6.60 6.43 7.9516.10 14.23 *Group 1: Açai fruit pulp (2,000 mg/kg, po.); **One dayprior to treatment

The individual body weights of female test subjects observed in the14-day post-treatment observation period is summarized below in Table48.

TABLE 48 FEMALES Group Body weights [g] Animal Day of code Day ofrandom- *Group 1: arrival ization Day −1** Day 2 Day 8 Day 15 861 141.0190.2 170.5 183.1 228.3 237.8 862 142.4 184.5 168.3 187.1 231.5 236.4863 143.1 181.9 165.6 182.0 210.7 225.9 864 149.7 179.9 164.3 184.6203.1 237.3 865 156.2 177.9 160.6 182.5 193.8 235.7 866 149.7 177.2156.0 177.5 189.4 227.4 867 154.2 174.3 158.8 174.6 191.1 222.8 868161.4 171.9 156.5 180.2 191.1 235.8 869 140.9 171.1 158.4 178.4 193.6222.6 870 140.5 166.8 153.2 174.1 193.2 245.9 Group 10 10 10 10 10 10size: Mean: 148.9 177.6 161.2 180.4 202.6 232.8 ±S.D.: 7.08 6.93 4.714.25 15.80 7.66 *Group 1: Açai fruit pulp (2,000 mg/kg, po.); **One dayprior to treatment

The individual body weight changes of male test subjects observed in the14-day post-treatment observation period is summarized below in Table49.

TABLE 49 MALES Body weight changes [g]** Animal code* Day 1 Days 2-7Days 8-14 851 26.4 73.0 26.8 852 23.6 49.9 48.5 853 25.7 57.5 50.0 85424.4 57.0 61.5 855 6.5 59.0 55.0 856 22.5 42.2 66.5 857 19.3 57.8 31.1858 18.0 69.1 21.2 859 21.6 34.8 84.2 860 23.7 50.8 50.1 Group size: 1010 10 Mean: 23.2 55.1 49.5 ±S.D.: 2:88 11.41 19.22 *Group 1: Açai fruitpulp (2,000 mg/kg, po.); **Differences calculated from body weightsweighed on Days 1 and 2, Days 2 and 8 as well as Days 8 and 15,respectively.

The individual body weight changes of female test subjects observed inthe 14-day post-treatment observation period is summarized below inTable 50.

TABLE 50 FEMALES Body weight changes [g]** Animal code* Day 1 Days 2-7Days 8-14 861 12.6 45.2 9.5 862 18.8 44.4 4.9 863 16.4 28.7 15.2 86420.3 18.5 34.2 865 21.9 11.3 41.9 866 21.5 11.9 38.0 867 15.8 16.5 31.7868 23.7 10.9 44.7 869 20.0 15.2 29.0 870 20.9 19.1 52.7 Group size: 1010 10 Mean: 19.2 22.2 30.2 ±S.D.: 3.34 13.00 15.73 *Group 1: Açai fruitpulp (2,000 mg/kg, po.); **(1) Differences calculated from body weightsweighed on Days 1 and 2, Days 2 and 8 as well as Days 8 and 15,respectively. (2) The body weight and the body weight gain of theanimals corresponded to their species and age throughout the study.

D. Gross Pathology

The gross pathology findings for test animals are summarized in Table51.

TABLE 51 Group 1 - MALES Group 2 - FEMALES Gross pathologyfinding/number of animals Treatment External* Internal** ExternalInternal Açai fruit pulp*** 0/10 0/10 0/10 0/10 *External: Animal ofaverage development. Skin, fur, visible mucous membranes are intact;**Internal: organs are without pathological changes; ***2,000 mg/kg, po.

The gross pathology findings for male test animals are summarized inTable 52.

TABLE 52 MALES Group Day 15 Animal code* External Internal 851 NoFinding** No Finding 852 No Finding No Finding 853 No Finding No Finding854 No Finding No Finding 855 No Finding No Finding 856 No Finding NoFinding 857 No Finding No Finding 858 No Finding No Finding 859 NoFinding No Finding 860 No Finding No Finding *Açai fruit pulp 2,000mg/kg, po.; **“No Finding” means: External: Animal of averagedevelopment. Skin, fur, visible mucous membranes are intact; Internal:organs are without pathological changes.

All animals survived until the scheduled autopsy, on day 15 and allproved to be free of toxic pathological changes.

E. Evaluation

No death occurred after single oral application of 2,000 mg/kg ‘Açaifruit pulp—Freeze dried’ dose. No toxic clinical symptoms occurred.Scheduled autopsy at day 15 revealed no toxic gross pathologicalchanges. It was concluded that no adverse effects were noted at singleoral dose of 2,000 mg/kg ‘Açai fruit pulp—Freeze dried’ in male andfemale rats.

Example 35

Acute Oral Toxicity Study of Jucara Fruit Pulp ‘Freeze-Dried’ with14-Day Posttreatment Observation Period in the Rat (Limit Test)

Studies were conducted to assess the acute oral toxicity of freeze-driedJucara fruit pulp with a 14-day posttreatment observation period in therat (limit test) ((Study code: PCDL-0222; Pharmaceutical Control andDevelopment Laboratory Co. Ltd., 9. Mexikoi Street Budapest, H-149).

I. General Information:

A. Dosage

Single oral limit dose of 2,000 mg/kg body weight of ‘Jucara fruitpulp—Freeze dried’ (Lot number: 2208) was applied to rats orally bygavage. Animals were observed for lethality and toxic symptoms for 14days. Gross pathological examination was carried out on the 15th day.The body weight of the animals corresponded to their species and agethroughout the study. No death occurred after oral administration of‘Jucara fruit pulp—Freeze dried’ at 2,000 mg/kg dose. No toxic clinicalsymptoms were observed. Scheduled autopsy carried out on day 15 revealedno toxic gross pathological changes. It was concluded that no adverseeffects were noted at single oral dose of 2,000 mg/kg ‘Jucara fruitpulp—Freeze dried’ in male and female rats.

B. Objective

To develop data on the potential toxicological effects of single oraladministration of Jucara fruit pulp—Freeze-dried in the rat. The testarticle is expected to use as dietary supplement.

C. Type of the Study

Preclinical toxicological study in compliance with the principles of theGood Laboratory Practice Regulations for Nonclinical Laboratory Studiesof the United States Food and Drug Administration and the Hungarian Act1998: XXVIII regulating animal protection. Limit test.

D. Deviations from the Study Protocol

i. Characteristics of Substance T 61 Used for ExterminationManufacturer:

Original protocol: Hoechst Veterinar GmbH

Final Report: Internet International

Reason: The name of the manufacturer has been changed.

ii. Mortality

Original protocol: Observations are made for 4 hours following treatmentand twice daily thereafter.

Final Report: Observations were made for 4 hours following treatment andtwice daily thereafter at the beginning and at the end of the workingday as well as once at weekends, until the morning of the 15th day.

Reason: Procedures have been described more precisely than originally

iii. General state, external appearance, behavior, and clinical symptoms

Original protocol: During the post-treatment period, animals are checkeddaily twice until the morning of the 15th day.

Final Report: During the post-treatment period, animals were checkeddaily twice until the morning of the 15th day except for weekends whenanimals were checked once.

Reason: Procedures have been described more precisely than originally

II. Test and Reference Articles

A. Characteristics of the Test Article

The characteristics of the test article are detailed below in Table 53.

TABLE 53 Name of the article: Jucara fruit pulp - Freeze dried Botanicalname: Euterpe edulis, Family: Palmae Plant part used: Fresh Frozen FruitPulp Manufacturer: Greater Continents do Brasil Ltda. Rua Alabastro,55-112, Aclimacäo 01531-010 Säo Paulo, SP Brasil Lot #: 2208Identification number 2002/22886 in PCDL: Residual moisture: max. <2%Physical characteristics: dark purple granular freeze dried powder withcharacteristic odor and flavor, hygroscopic Storage conditions:refrigerated according to USP (2-8° C., humidity not controlled),re-sealed quickly if opened

B. Microbiological Analysis

Microbiological limit test according to c. USP was carried out by theMicrobiological Department of PCDL.

C. Characteristics of the Article Used for Suspending the Test Article

i. Methylcellulose

Methylcellulose (Bach No. 127H1066; Expiration February 2003) wascommercially obtained from Sigma and stored at room temperature prior touse.

ii. Distilled Water

Distilled water (Batch No. A0010102; Expiration March 2003) wascommercially obtained from PCDL and stored at room temperature prior touse.

iii. Characteristics of Article Used for Overanesthesia Before Necropsy

T 61 (Batch No. 09W008; Expiration May 2006) containing 0.2 gembutramide, 0.005 g tetracaine hydrochloride, and 0.05 g mebezoniumiodide per ml was commercially obtained from Intervet International andstored at room temperature, in a safe box for poisonous drugs prior touse.

iv. Formulation of the Test Article

The necessary amount of the test article was weighed and suspended in 1%methylcellulose containing solution not earlier than 30 min beforeadministration.

The following suspension was prepared: Nominal dose 2000 mg/kg: 5.0 gJucara fruit pulp ad 50 ml of 1% methylcellulose solution. Thesuspension was then stirred during treatment with a Radelkis magneticstirrer type OP-951.

v. Concentration Control of the Formulated Test Article

Samples of the formulated test substance were taken for check of theconcentration and homogeneity. Concentration and homogeneity check wasperformed by gravimetry.

The concentration of all three samples measured in triplicates of theupper, intermediate, and lower parts of the suspension (homogeneitycheck) were within the acceptable ±10% limits i.e., upper: +9.4±4.2%,intermediate: +9.4±4.6%, lower: +6.6±2.0%.

III. Test System

A. Animals

Sprague Dawley rat, Crl:CD BR (6-7 weeks of age at arrival) were used inthe present studies. The males had body weights that ranged from 143.8 gto 151.9 g. The females had body weights that ranged from 144.2 g to161.6 g. A pool of animals ordered: 30 (15 males, 15 females). Number ofanimals involved in the study: 20 (10 males, 10 females). Rats werecommercially obtained from Charles River Hungary Ltd. Animals were SPFat arrival and kept in a conventional environment during the study. Therat is commonly used for toxicological studies in accordance withinternational recommendations. The Sprague Dawley strain is a well-knownlaboratory model with sufficient historical data.

The animals were identified by ear numbering technique and housed incages by five of the same sex. The cages were labeled with tagsindicating the I.D. numbers of the rats, the study code, the groupidentification, route of administration, sex and the starting and endingdates of the experimental period.

The animal housing conditions are summarized below in Table 54.

TABLE 54 Hygienic level: conventional Type of animal cages: type IImacrolone Size of cage: H × W × D: 17.5 cm × 22.5 cm × 37.5 cm Cleaning:by changing the bottom of the cages three times a week Number of animalsper cage:  5 Number of animal keeping room: 123

The environmental conditions are summarized below in Table 55.

TABLE 55 Air exchange: approximately 15 times/hour Temperature: 22 ± 3°C. Relative humidity: 30-70% Lighting: artificial, 12 hour light-darkcycles.

The temperature and the relative humidity were continuously recorded.

The animals were given free access to standardized rat and mouse dietVRF-1 except for the overnight fasting period prior to treatment, duringthe treatment and for the two first hours of the posttreatmentobservation. The composition of the diet was controlled by theManufacturer Altromin GmbH, D-4937 Lage/Lippe Lange Str. 42. The dietwas identified by the date of manufacturing (30 Sep. 2002), stability: 4months. Rats had free access to tap water via drinking bottles. Drinkingwater is checked monthly by the Microbiological Department of PCDL. Theanimals were observed for 5 days prior to the treatment. Only healthyanimals, free from any clinical symptom were used in the study.

Grouping of the animals was made with a random table generated by acomputer. The animals were randomly assigned to groups on the basis oftheir body weight, so that the distribution of the body weights in theindividual groups were similar.

IV. Experimental Design

The dose levels and group division are summarized below in Table 56.

TABLE 56 Number of Identification Group Dose Animals numbers NumberTreatment mg/kg Males Females Males Females 1 Jucara fruit 2,000 10 —871-880 — pulp 2 Jucara fruit 2,000 — 10 — 881-890 pulp

The rational for the dose selection is as follows. The expected humandaily dose of Jucara fruit pulp is approx. 1000 mg per day whichcorresponds to 14 mg/kg body weight of an adult (70 kg) or 50 mg/kg fora 4 years old child (20 kg). The 2000 mg/kg limit dose applied in thisstudy corresponds to 140 times of the daily dose if consumed by an adultor 40 times of it if 5 g is calculated for a child's body weight.

V. Administration

Application was oral by gavage. The route of application was selected incompliance with international guidelines. The oral route is theanticipated route of human exposure to the test article. The applicationof the test article was given in a single dose. The test article wasadministered in a volume of 20 ml/kg body weight. The experimentalperiod consisted of 5 days of acclimatization, treatments day, 14 dayspost-treatment observation period including the treatment's day, and the15th day: necropsy.

VI. Observations, Examinations

A. Lethality

Observations were made for 4 hours following treatment and twice dailythereafter at the beginning and at the end of working days as well asonce at weekends until the morning of the 15th day. The time of deathshould have been recorded as accurately as possible.

B. General State, External Appearance, Behavior, and Clinical Symptoms

Careful clinical observation of the rats was carried out once before theexposure, then, after the treatment for 6 hours continuously. During thesubsequent period, animals were checked daily twice until the morning ofthe 15th day except for weekends, when animals were checked once. Signsto be observed included changes in skin, fur, eyes and visible mucousmembranes; occurrence of secretions and excretions and autonomicactivity (e.g., lacrimation, piloerection, diarrhea, pupil size, unusualrespiratory pattern). Furthermore, potential changes in gait, postureand response to handling as well as the presence of somnolence,trembling, clonic or tonic movements, stereotypes or bizarre behaviorwere recorded.

C. Body Weight

Animals were weighed at arrival in the laboratory, on the day ofrandomization, on the day of treatment, as well as on the 2nd, 8th, and15th day of the experiment prior to the necropsy.

VII. Necropsy and Histological Examination

A. Necropsy

All surviving rats on completion of the posttreatment observation periodwere exterminated under T61 overanaesthesia and autopsied. External andinternal status were carefully observed and recorded. No microscopicexamination of organs was performed.

VIII. Evaluation, Statistical Analysis

Groups of males and females were evaluated separately.

A. Parametric Values

Mean values and standard deviations were calculated of the body weights.

B. Non Parametric Values (Lethality and Clinical Symptoms)

The incidence of lethality, clinical symptoms, and gross findings weretabulated.

IX. Procedures

The experiments were performed according to the current StandardOperating Procedures of the Department of Toxicology of thePharmaceutical Control and Development Laboratory Co. Ltd.

X. Animal Protection

In the interests of animal welfare the unnecessary use of animals wasavoided. To order the mild extermination of unambiguously moribundanimals was the responsibility of the study director. The present method(limit test) uses a reduced number of experimental animals in comparisonto other known and acknowledged acute toxicity tests.

XI. Data Recording and Archivation

All original data are maintained, as dictated by the Standard OperatingProcedures, on appropriate forms as follows:

Test Compound weighing

Animal room logbook

Body weight logbooks

Lethality and Clinical observations logbooks

Postmortem records

The data obtained in the course of the study were collected in a StudyFile. The Study Protocol, all data generated during and as a result ofthe study, the documents and all information in connection with thestudy, a control sample of the test article and the Final Report will bestored at least for 15 years in the Archives of the PCDL then offered tothe Sponsor.

XII. Results

A. Lethality

The lethality observed in the 14-day post-treatment observation periodis summarized below in Table 57.

TABLE 57 Group 1 - MALES Group 2 - FEMALES Treatment death/number ofanimals Juçara fruit pulp; 0/10 0/10 2,000 mg/kg, po.

Table 58 summarizes the individual lethality data for male test subjectsin the acute oral toxicity study of ‘Jucara fruit pulp—Freeze dried’with 14-day posttreatment observation period in the rat (limit test).

TABLE 58 MALES Group/ DAYS OF OBSERVATION PERIOD Animal Day Day Day DayDay Day Day Code 1* Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 1011 12 13 14 15 871 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 872 0 0 0 0 0 0 0 0 0 00 0 0 0 0 873 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 874 0 0 0 0 0 0 0 0 0 0 0 00 0 0 875 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 876 0 0 0 0 0 0 0 0 0 0 0 0 0 00 877 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 878 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0879 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 880 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0*Açai fruit pulp (2,000 mg/kg, po.); Remark: 0 = No Lethality; *Day 1 =Treatment's day

Table 59 summarizes the individual lethality data for female testsubjects in the acute oral toxicity study of ‘Jucara fruit pulp—Freezedried’ with 14-day posttreatment observation period in the rat (limittest).

TABLE 59 FEMALES Group* DAYS OF OBSERVATION PERIOD Animal Day Day DayDay Day Day Day Code 1* Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 910 11 12 13 14 15 881 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 882 0 0 0 0 0 0 0 00 0 0 0 0 0 0 883 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 884 0 0 0 0 0 0 0 0 0 00 0 0 0 0 885 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 886 0 0 0 0 0 0 0 0 0 0 0 00 0 0 887 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 888 0 0 0 0 0 0 0 0 0 0 0 0 0 00 889 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 890 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0*Açai fruit pulp (2,000 mg/kg, po.); Remark: 0 = No Lethality; *Day 1 =Treatment's day

No death occurred following the single oral administration of 2,000mg/kg dose of ‘Jucara fruit pulp—Freeze dried’ to rats. All males andfemales survived until the end of the 14-day observation period.

B. Clinical Symptoms

The clinical symptoms observed in the 14-day post-treatment observationperiod are summarized below in Table 60.

TABLE 60 Group 1 - MALES Group 2 - FEMALES Treatment death/number ofanimals Juçara fruit pulp; 0/10 0/10 2,000 mg/kg, po.

Table 61 summarizes the individual clinical symptoms for male testsubjects in the acute oral toxicity study of ‘Jucara fruit pulp—Freezedried’ with 14-day posttreatment observation period in the rat (limittest).

TABLE 61 MALES Group* DAYS OF OBSERVATION PERIOD Animal Day Day Day DayDay Day Day Code 1* Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 1011 12 13 14 15 871 SF SF SF SF SF SF SF SF SF SF SF SF SF SF SF 872 SFSF SF SF SF SF SF SF SF SF SF SF SF SF SF 873 SF SF SF SF SF SF SF SF SFSF SF SF SF SF SF 874 SF SF SF SF SF SF SF SF SF SF SF SF SF SF SF 875SF SF SF SF SF SF SF SF SF SF SF SF SF SF SF 876 SF SF SF SF SF SF SF SFSF SF SF SF SF SF SF 877 SF SF SF SF SF SF SF SF SF SF SF SF SF SF SF878 SF SF SF SF SF SF SF SF SF SF SF SF SF SF SF 879 SF SF SF SF SF SFSF SF SF SF SF SF SF SF SF 880 SF SF SF SF SF SF SF SF SF SF SF SF SF SFSF *Açai fruit pulp (2,000 mg/kg, po.); Remarks: SF = Symptom Free; *Day1 = Treatment's day

Table 62 summarizes the individual clinical symptoms for female testsubjects in the acute oral toxicity study of ‘Jucara fruit pulp—Freezedried’ with 14-day posttreatment observation period in the rat (limittest).

TABLE 62 FEMALES Group* DAYS OF OBSERVATION PERIOD Animal Day Day DayDay Day Day Day Code 1* Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 910 11 12 13 14 15 881 SF SF SF SF SF SF SF SF SF SF SF SF SF SF SF 882SF SF SF SF SF SF SF SF SF SF SF SF SF SF SF 883 SF SF SF SF SF SF SF SFSF SF SF SF SF SF SF 884 SF SF SF SF SF SF SF SF SF SF SF SF SF SF SF885 SF SF SF SF SF SF SF SF SF SF SF SF SF SF SF 886 SF SF SF SF SF SFSF SF SF SF SF SF SF SF SF 887 SF SF SF SF SF SF SF SF SF SF SF SF SF SFSF 888 SF SF SF SF SF SF SF SF SF SF SF SF SF SF SF 889 SF SF SF SF SFSF SF SF SF SF SF SF SF SF SF 890 SF SF SF SF SF SF SF SF SF SF SF SF SFSF SF *Açai fruit pulp (2,000 mg/kg, po.); Remarks: SF = Symptom Free;*Day 1 = Treatment's day

No toxic symptoms were observed on the day of application and during the14-day posttreatment period at any group of the treated animals.

C. Body Weights

The body weights of male test subjects observed in the 14-daypost-treatment observation period is summarized below in Table 63.

TABLE 63 MALES Body weights [g] Day of Group 1 Day of random Treatment*arrival ization Day −1 Day 2 Day 8 Day 15 Group 10 10 10 10 10 10 size:Mean: 148.1 198.6 181.8 195.6 255.5 313.8 ±S.D.: 3.37 6.33 6.84 14.978.98 19.09 *Juçara fruit pulp (2,000 mg/kg, p.o.); ** One day prior totreatment

The body weights of female test subjects observed in the 14-daypost-treatment observation period is summarized below in Table 64.

TABLE 64 FEMALES Body weights [g] Day of Group 1 Day of random Treatmentarrival ization Day −1** Day 2 Day 8 Day 15 Group 10 10 10 10 10 10size: Mean: 152.6 177.8 162.7 279.9 204.9 227.2 ±S.D.: 6.25 7.46 7.139.61 5.81 19.25 Juçara fruit pulp (2,000 mg/kg, p.o.); ** One day priorto treatment

The body weight changes of male test subjects observed in the 14-daypost-treatment observation period is summarized below in Table 65.

TABLE 65 MALES Body weight changes [g] Groups* Day 1 Day 2 Day 8 Groupsize: 10 10 10 Mean: 13.8 60.0 58.3 ±S.D.: 10.76 10.28 20.36 *Juçarafruit pulp; 2,000 mg/kg, po.

The body weight changes of female test subjects observed in the 14-daypost-treatment observation period is summarized below in Table 66.

TABLE 66 FEMALES Body weight changes [g] Groups* Day 1 Days 2-7 Days8-14 Group size: 10 10 10 Mean: 16.9 25.4 22.3 ±S.D.: 4.92 5.62 14.59*Juçara fruit pulp; 2,000 mg/kg, po.

The individual body weights of mate test subjects observed in the 14-daypost-treatment observation period is summarized below in Table 67.

TABLE 67 MALES Body weights [g] Group* Day of Animal Day of random- codearrival ization Day −1** Day 2 Day 8 Day 15 871 146.3 208.2 191.5 213.4265.7 328.7 872 151.9 205.3 185.1 208.7 255.1 314.2 873 147.9 202.5188.8 209.3 265.9 310.5 874 143.8 202.2 178.5 204.7 263.5 323.5 875148.9 200.1 180.3 207.1 261.5 332.7 876 145.6 198.9 178.3 181.4 258.8284.3 877 153.9 196.2 188.3 194.0 248.8 322.0 878 146.9 192.6 183.3186.7 242.9 334.9 879 144.3 190.7 171.3 174.0 250.8 279.6 880 151.2189.2 172.5 176.2 242.2 307.3 Group 10 10 10 10 10 10 size: Mean: 148.1198.6 181.8 195.6 255.5 313.8 ±S.D.: 3.37 6.33 6.84 14.97 8.98 19.09*Juçara fruit pulp (2,000 mg/kg, po); **One day prior to treatment

The individual body weights of female test subjects observed in the14-day post-treatment observation period is summarized below in Table68.

TABLE 68 FEMALES Body weights [g] Group* Day of Animal Day of random-code arrival ization Day −1** Day 2 Day 8 Day 15 881 154.2 193.4 174.4197.1 216.0 263.8 882 159.9 185.6 175.0 187.4 21L4 256.3 883 144.2 182.0167.0 184.4 204.0 237.1 884 161.6 178.1 162.9 185.4 206.5 223.1 885158.6 178.1 160.0 185.1 207.2 225.5 886 150.3 175.9 159.4 173.4 197.0208.9 887 155.7 172.3 156.3 167.6 200.4 216.2 888 148.8 171.4 158.6174.1 200.2 211.5 889 147.5 170.7 157.5 169.7 200.2 218.1 890 145.3170.5 155.8 171.5 206.5 211.5 Group 10 10 10 10 10 10 size: Mean: 152.6177.8 162.7 179.6 204.9 227.2 ±S.D.: 6.25 7.46 7.13 9.61 5.81 19.25*Juçara fruit pulp (2,000 mg/kg, po); ** One day prior to treatment

The individual body weight changes of male test subjects observed in the14-day post-treatment observation period is summarized below in Table69.

TABLE 69 MALES Groups* Body weight changes [g]** Animal code Day 1 Days2-7 Days 8-14 871 21.9 52.3 63.0 872 23.6 46.4 59.1 873 20.5 56.6 44.6874 26.2 58.8 60.0 875 26.8 54.4 71.2 876 3.1 77.4 25.5 877 5.7 54.873.2 878 3.4 56.2 92.0 879 2.7 76.8 28.8 880 3.7 66.0 65.1 Group size:10 10 10 Mean: 13.8 60.0 58.3 ±S.D.: 10.76 10.28 20.36 *Juçara fruitpulp (2,000 mg/kg, p.o.); **Differences calculated from body weightsweighed on Days 1 and 2, Days 2 and 8 as well as Days 8 and 15,respectively.

The individual body weight changes of female test subjects observed inthe 14-day post-treatment observation period is summarized below inTable 70.

TABLE 70 FEMALES Groups* Body weight changes [g]** Animal code Day 1Days 2-7 Days 8-14 881 22.7 18.9 47.8 882 12.4 240 44.9 883 17.4 19.633.1 884 22.5 21.1 16.6 885 25.1 22.1 18.3 886 14.0 23.6 11.9 887 11.332.8 15.8 888 15.5 26.1 11.3 889 12.2 30.5 17.9 890 15.7 35.0 5.0 Groupsize: 10 10 10 Mean: 16.9 25.4 22.3 ±S.D.: 4.92 5.62 14.59 *Juçara fruitpulp (2,000 mg/kg, p.o.); **Differences calculated from body weightsweighed on Days 1 and 2, Days 2 and 8 as well as Days 8 and 15,respectively.

The body weight and the body weight gain of the animals corresponded totheir species and age throughout the study.

D. Gross Pathology

The gross pathology findings for test animals are summarized in Table71.

TABLE 71 Group 1 - MALES Group 2 - FEMALES External Internal ExternalInternal Treatment finding/number of animals Juçara fruit pulp; 0/100/10 0/10 0/10 2,000 mg/kg, po.

The gross findings for male test animals is summarized in Table 72.

TABLE 72 MALES Groups* Day 15 Animal code External Internal 871 NoFinding** No Finding 872 No Finding No Finding 873 No Finding No Finding874 No Finding No Finding 875 No Finding No Finding 876 No Finding NoFinding 877 No Finding No Finding 878 No Finding No Finding 879 NoFinding No Finding 880 No Finding No Finding *Juçara fruit pulp; (2,000mg/kg, po.); **“No Finding” here means the following: External: Animalof average development. Skin, fur, visible mucous membranes are intact;Internal: organs are without pathological changes

The gross findings for female test animals is summarized in Table 73.

TABLE 73 FEMALES Groups* Day 15 Animal code External Internal 881 NoFinding** No Finding 882 No Finding No Finding 883 No Finding No Finding884 No Finding No Finding 885 No Finding No Finding 886 No Finding NoFinding 887 No Finding No Finding 888 No Finding No Finding 889 NoFinding No Finding 890 No Finding No Finding *Juçara fruit pulp; (2,000mg/kg, po.); **“No Finding” here means the following: External: Animalof average development. Skin, fur, visible mucous membranes are intact;Internal: organs are without pathological changes

All animals survived until the scheduled autopsy, on day 15 and allproved to be free of toxic pathological changes.

E. Evaluation

No death occurred after single oral application of 2,000 mg/kg ‘Jucarafruit pulp—Freeze dried’ dose. No toxic clinical symptoms occurred.Scheduled autopsy at day 15 revealed no toxic gross pathologicalchanges. It was concluded that no adverse effects were noted at singleoral dose of 2,000 mg/kg ‘Jucara fruit pulp—Freeze dried’ in male andfemale rats.

Equivalents

While the apparatus and method have been described in terms of what arepresently considered to be the most practical and preferred embodiments,it is to be understood that the disclosure need not be limited to thedisclosed embodiments. It is intended to cover various modifications andsimilar arrangements included within the spirit and scope of the claims,the scope of which should be accorded the broadest interpretation so asto encompass all such modifications and similar structures. The presentdisclosure includes any and all embodiments of the following claims.

1. A method of inhibiting cyclooxygenase enzyme activity in a mammal,the method comprising administering to the mammal an effective amount ofa dietary supplement comprising a freeze-dried Jucara fruit pulpcomposition, wherein the composition comprises: (a) a total anthocyaninconcentration greater than about 1 milligram per gram total weight; (b)an ORAC_(FL) value greater than about 350 micromole TE per gram totalweight; and (c) a residual water content less than about 3 weightpercent of the total weight, wherein the composition quenches freeradicals and reduces the damage induced by pathological free radicals;or an effective amount of a freeze-dried Jucara fruit pulp composition,wherein the composition comprises a cyclooxygenase inhibition valuegreater than about 15 Aspirin® mg equivalent per gram total weight; anda residual water content less than about 3 weight percent of the totalweight.
 2. The method of claim 1, wherein the composition furthercomprises a pharmaceutically acceptable carrier.
 3. The method of claim1, wherein the composition is administered by a route of administrationselected from the group consisting of: oral, intravenous,intraperitoneal, subcutaneous, intramuscular, intraarticular,intraarterial, intracerebral, intracerebellar, intrabronchial,intrathecal, topical, and aerosol route.
 4. A method of treating adisease or an injury associated with increased cyclooxygenase enzymeactivity in a mammal, the method comprising administering to the mammalan effective amount of a dietary supplement comprising a freeze-driedJucara fruit pulp composition, wherein the composition comprises: (a) atotal anthocyanin concentration greater than about 1 milligram per gramtotal weight; (b) an ORAC_(FL) value greater than about 350 micromole TEper gram total weight; and (c) a residual water content less than about3 weight percent of the total weight, wherein the composition quenchesfree radicals and reduces the damage induced by pathological freeradicals; or an effective amount of a freeze-dried Jucara fruit pulpcomposition, wherein the composition comprises a cyclooxygenaseinhibition value greater than about 15 Aspirin® mg equivalent per gramtotal weight; and a residual water content less than about 3 weightpercent of the total weight.
 5. The method of claim 4, wherein thecomposition further comprises a pharmaceutically acceptable carrier. 6.The method of claim 4, wherein the composition is administered by aroute of administration selected from the group consisting of: oral,intravenous, intraperitoneal, subcutaneous, intramuscular,intraarticular, intraarterial, intracerebral, intracerebellar,intrabronchial, intrathecal, topical, and aerosol route.
 7. The methodof claim 4, wherein the disease or injury is selected from the groupconsisting of: cancer, colon cancer, breast cancer, inflammatory boweldisease, Crohn's disease, vascular disease, arthritis, ulcer, acuterespiratory distress syndrome, ischemia-reperfusion injury,neurodegenerative disorders, autism, Parkinson's Disease, Alzheimer'sDisease, gastrointestinal disease, tissue injury induced byinflammation, and tissue injury induced by an environmental toxin.